(Received for publication, June 26, 1995; and in revised form, August 30, 1995)
From the
The non-heme iron-dependent metalloenzyme, rat hepatic
phenylalanine hydroxylase (EC 1.14.16.1; phenylalanine 4-monooxygenase
(PAH)) was overexpressed in Escherichia coli and purified to
homogeneity, allowing a detailed comparison of the kinetic,
hydrodynamic, and spectroscopic properties of its allosteric states.
The homotetrameric recombinant enzyme, which is highly active and
contains 0.7-0.8 iron atoms per subunit, is identical to the
native enzyme in several properties: K,
6-methyltetrahydropterin = 61 µM and L-Phe
= 170 µM; V
= 9
s
(compared to 45 µM, 180
µM, and 13 s
for the rat hepatic
enzyme). L-Phe and lysolecithin treatment induce the
rPAH
rPAH
(where r is recombinant)
allosteric transformation necessary for rPAH activity. Characteristic
changes in the fluorescence spectra, increased hydrophobicity, a large
activation energy barrier, and a 10% volume increase of the tetrameric
structure are consistent with a significant reorganization of the
protein following allosteric activation. However, optical and EPR
spectroscopic data suggest that only minor changes occur in the primary
coordination sphere (carboxylate/histidine/water) of the catalytic iron
center. Detailed steady state kinetic investigations, using
6-methyltetrahydropterin as cofactor and lysolecithin as activator,
indicate rPAH follows a sequential mechanism. A catalytic Arrhenius E
of 14.6 ± 0.3 kcal/mol subunit was
determined from temperature-dependent stopped-flow kinetics data. rPAH
inactivates during L-Phe hydroxylation with a half-life of 4.3
min at 25 °C, corresponding to an Arrhenius E
of 10 ± 1 kcal/mol subunit for the inactivation process.
Catechol binding (2.4
10
M
) is shown to occur only at catalytically
competent iron sites. Ferrous rPAH binds NO, giving rise to an S
= 3/2 spin system.
Phenylketonuria (PKU) ()is a relatively common inborn
error of amino acid metabolism caused by the accumulation of
neurotoxins derived from L-phenylalanine. A major clinical
manifestation of untreated PKU is progressive postnatal brain damage
leading to severe mental retardation that is thought to arise from the
decreased protection of myelin proteins against proteolytic
degradation. This degenerative process may be slowed in newborns only
through the immediate and rigorous implementation of a controlled L-Phe diet. The Mendelian inheritance pattern of this
degenerative condition indicates there is a single detoxifying enzyme
absent in phenylketonurics. Phenylketonuric liver biopsies show
severely reduced or absent phenylalanine hydroxylase (EC 1.14.16.1; L-phenylalanine tetrahydropterin:oxygen oxidoreductase (PAH))
activity, which ordinarily regulates blood [L-Phe]
and prevents PKU. Worldwide screening efforts have allowed the
identification of over 150 PAH mutations that cause PKU(1) .
Mammalian PAH is a soluble, homotetrameric protein (molecular mass
= 51.7 kDa) whose activity depends upon the presence of a single
non-heme iron center per subunit(2) . Rat hepatic PAH is the
most extensively studied of the tetrahydrobiopterin
(BH)-dependent amino acid
hydroxylases(3, 4) , a highly homologous family of
enzymes that includes TyrH and TrpH. These brain enzymes perform the
rate-limiting steps in the biosynthesis of catecholamine
neurotransmitters and serotonin, respectively. All of the
pterin-dependent hydroxylases are thought to contain similar active
sites, due to the very high conservation of ``catalytic
domain'' residues, as well as their similar substrates, common
biopterin cofactor, and shared sensitivity to potentially chelating
inhibitors(3, 5, 6) . The highest level of
DNA sequence homology among these enzymes, as well as the location of
many of the more severe PKU mutations, occurs in PAH exon 7, which may
be of particular importance because it is a constituent of the
catalytic core.
PAH interacts in a dual fashion with each of its
substrates; in addition to their roles in the catalytic cycle, L-Phe is the obligate allosteric activator of PAH, while
reduction of resting state ferric PAH to the ferrous state is performed
by BH(7, 8) . Formation of reduced,
phenylalanine-activated PAH is concurrent with the appearance of a
catalytically competent species(3) .
Activation by L-Phe is typical of a catabolic enzyme's feed-forward
response to substrate (Fig. 1). The slow conversion of low
activity, T state PAH (PAH) to R state PAH
(PAH
[L-Phe]) is facilitated by
phosphorylation and inhibited by
BH
(9, 10, 11) . PAH is isolated
from liver in a partially phosphorylated state, due to regulation by
phosphorylation/dephosphorylation at Ser-16(12) . The amino
acid substrate specificity is diminished following substrate activation
or limited proteolysis (exposing a catalytic core) but not by
phosphorylation. Under these conditions, for example, PAH will
hydroxylate tryptophan at the same 5-position as
TrpH(13, 14) .
Figure 1:
Catalytic scheme for
PAH. PAH = resting/T state PAH, PAH
= activated/R state PAH, and PAH
= inactivated PAH; k
, k
, and k
are all
apparent first-order rate constants for the allosteric activation,
irreversible inactivation, and catalytic processes. The obligate
prereduction of PAH has been omitted for clarity; only
{Fe
} PAH
is catalytically
active. The NIH shift of para-X to meta-X is known to occur when X =
H,
H, chlorine, bromine, or CH
.
Also shown is the recycling of the initial 4a-hydroxypterin product
back to reduced tetrahydropterin. In reaction 1, a spontaneous
dehydration reaction that is accelerated in liver by 4a-carbinolamine
dehydratase/DCoH, q-6-MPH
is formed. In reaction 2 this unstable oxidized form of the pterin is
reduced, either by dihydropteridine reductase/NADPH (in liver and in
the Nielsen/Kaufman assay) (92, 95) or by DTT (Shiman
assay)(31) .
The PAH active site is of additional interest in that it is able to perform the same spectrum of chemical transformations as cytochrome P450 without benefit of a heme prosthetic group(15, 16, 17) . A variety of other transformations of isosteric amino acids is possible, including epoxidation, aliphatic hydroxylation, and the formation of nitrogen and sulfur oxides(15, 16, 17) . In contrast to the microsomal hydroxylases, PAH activity is tightly regulated at the post-translational as well as transcriptional level, and its oxidative chemistry is much more specific.
The molecular genetics of phenylalanine hydroxylase have been of intense interest ever since the human gene was located (18) because of the molecular nature of PKU, the possibility of preparing vectors for gene therapy(19) , and the ability to prepare PKU-mutant proteins in an effort to link genotype and phenotype(20) . Using heterologous expression followed by activity assays of cell lysate, various PKU mutations have been associated with unstable mRNA, unstable/unfolded proteins, or apparently intact proteins that have low activity(21) . In the last case, the specific origin of the molecular defect in the complicated activation and catalytic pathways of PAH cannot be determined until the protein is purified to homogeneity and compared with a thoroughly characterized wild-type PAH. The dissection of this process into discrete, fully characterized steps and structures is essential for determining both the mechanism of catalysis and ultimately the chemical basis of PKU, but it has not yet been possible because of the heterogeneity of the protein. For instance, the observation of PKU due to diminished sensitivity to L-Phe might be due to either a decreased active site or allosteric site L-Phe affinity, or it might result from improper phosphorylation, subunit assembly, allosteric interactions, etc.
We have embarked upon a research program involving detailed spectroscopic and mechanistic characterization of PAH, with special attention to the active site iron's properties. No previous method of preparing PAH from rat liver tissue or a recombinant source is suitable for the purification of the large amounts (>100 mg) of spectroscopically homogeneous material that we require. PAH isolated from rat liver is a mixture of alleles, having variable levels of phosphate and different types of tightly bound iron(3) . Recombinant PAH expressed in baculovirus is fully phosphorylated and highly active but would be expensive to manufacture on a large scale(22) . Prokaryotic expression has the potential to provide sufficient quantities of enzyme but has not yet been optimized for yield, iron content, and purity or characterized in sufficient detail. To establish a base line from which we can reliably compare different states and forms of the enzyme, we report an efficient, large-scale expression of rat hepatic PAH in Escherichia coli and a comprehensive set of its kinetic and spectroscopic properties.
The
large (34 kcal/mol) (3) Arrhenius activation barrier to
the T
R conversion suggests that significant structural
distortions of the PAH tetramer occur during allosteric activation. It
is not known if the alterations are largely protein-centered or are
caused by a change in the coordination environment of the essential
active site iron. Using a combination of spectroscopic probes of this
process, we show that the direct effects activation has on the active
site configuration are limited, while prominent changes occur at the
tertiary/quaternary levels of protein structure. The steady state
properties of the R state active site, across an unprecedentedly broad
range of substrate concentrations, indicate sequential binding of L-Phe, 6-MPH
, and O
prior to catalytic
steps. We introduce a novel adduct of reduced PAH and NO, which models
the interaction of Fe
and O
at the active
site and can be used as a structural probe of that complex. A model
based on metal-centered, biophysical, and kinetic observations of
recombinant PAH is presented to explain the reactivity properties of
the enzyme in terms of the T
R transition.
Generally
the purifications do not include an iron-addition step. When this step
was performed, catalase (2,000 units/ml) and superoxide dismutase (10
units/ml) were added to the supernatant between the first spin and the L-Phe activation step. After 1 min at 25 °C, the lysate
was made 5 mM in DTT and 0.75 mM in
Fe(NH)
(SO
)
, followed by
the second spin and subsequent purification steps.
Determinations of L-Phe or lysolecithin activation at equilibrium were carried out as described(33) . Briefly, 20 µl at the indicated [activator], containing 10 µg of enzyme, was preincubated for 5 min at 25 °C and then added as the last ingredient to a standard assay mix at 11 °C. The lower temperature is necessary to minimize further activation by 1 mML-Phe. No significant variation in the initial slope was observed over the first 30 s of reaction, which was the interval used for velocity determinations.
For activity assays performed using
stopped-flow detection, the reagents are loaded into two syringes; the
first syringe contains rPAH (±L-Phe) in phosphate
buffer, and the second syringe contains pterin, DTT, and L-Phe
in a modification of a previously reported procedure(34) . The
solutions are preincubated 5 min at 25 °C, loaded into syringes,
and equilibrated at the desired assay temperature for 10 min before the
first of several injections. Stopped-flow assays show a lag in the
first few seconds of assay due to the obligate prereduction of rPAH,
which could be avoided by the addition of up to 20 equivalents of
6-MPH to the enzyme-containing syringe.
Observation of
the q-6-MPH formed by reduction of rPAH was
carried out at 334 nm, where the formation of q-6-MPH
results in an increased absorbance (at this wavelength, the molar
absorptivities of 6-MPH
and 6-MPH
are equal and
lower than that of q-6-MPH
). In these assays, DTT
is omitted, and pterin is added as the last ingredient to an otherwise
complete assay mix at pH 8.0 exactly as described. Under these
conditions, the 4a-carbinolamine form of 6-MPH
(4a-hydroxy-6-MPH
) that is the initial product of the catalytic cycle dehydrates slowly to q-6-MPH
, and so the initial product can be
identified for each reaction(7) .
and solved as a function of C for K and
(where C = concentration of catechol, E
= concentration of enzyme containing active iron, K = catechol
rPAH complex dissociation constant, and
= complex molar extinction coefficient). Steady state
kinetics double-reciprocal analysis was done using Mathematica (Wolfram
Research) using the sequential mechanism velocity expression:
where v = initial velocity, V =
maximum velocity, K and K
are the Michaelis and dissociation constants for species x, respectively. Data were weighted as the square of the
velocities(39) . Routine analyses were performed with
KaleidaGraph (Abelbeck Software). Analysis of transient kinetics was
carried out using the Kinsim/Fitsim software package by C. Frieden and
co-workers(40, 41) , rewritten by G. Mudunuri (Texas A
& M, College Station) for use on a Silicon Graphics workstation.
The kinetic mechanism used in simulations was that employed by Shiman
and Gray(33) , in which the L-Phe activation and iron
site inactivation are treated as sequential first-order processes.
Figure 2: A, purification of rPAH. Lanes 1-5 contain 20 µg of protein from the indicated purification steps (see Table 1). Lanes 5-7 contain 2 µg of protein. B, purification of rPAH to homogeneity. Each lane contains 2 µg of protein at the indicated stage of purification. Also shown is rPAH purified through the Mono Q step (see ``Experimental Procedures''). C, Western blot of 1 µg of protein, developed using a polyclonal antibody to PAH. The functionally similar ``W'' and ``L'' alleles can be distinguished in the rat hepatic PAH lane, whereas the rPAH shows only the ``W'' allele, as expected. Size standard positions are indicated.
Direct adaptation of the purification scheme of Shiman et al.(32) for the scale described above resulted in rPAH of activity 7 ± 1 units/mg; purification of protein from two combined 10-liter growths is described in Table 1. Recombinant PAH is isolated with specific activity and activation behavior essentially identical to that of enzyme from rat liver. The phenyl-Sepharose-based purification procedure requires that rPAH undergo a reversible, L-Phe-induced increase in hydrophobicity. This affords a purification from incorrectly assembled forms of PAH, some of which may have enzyme activity, thereby lowering the apparent recovery.
The
expression of the rat hepatic PAH cDNA in E. coli is
correlated with the appearance of a M =
51,000 protein that cross-reacts with a polyclonal antibody to rat
hepatic PAH in a Western blot. Only full-length protein is purified by
this procedure, despite the observation of small amounts of a
cross-reactive 47-kDa protein observed in soluble cell lysate (data not
shown). The electrophoretic mobility of rPAH is identical to that of
the rat ``W'' allele, from which the cDNA clone was obtained.
Under SDS-PAGE conditions, the minor ``I'' allele (Ile-371
rather than Thr-371) is resolved at a slightly smaller apparent M
(
49,000). Total amino acid analysis was
performed on the recombinant protein, which was found to agree well
with the predicted protein sequence. An extinction coefficient for rPAH
could be calculated upon this basis: a 1 mg/ml solution at 25 °C in
0.12 M phosphate, pH 6.8, gives rise to an absorbance of 1.05
at 280 nm and gives an apparent Bradford reading of 1.05 mg/ml.
The
relatively strong induction of rPAH (on the order of 5% total bacterial
protein) allowed the deletion of several items from the standard
purification, among them the use of Tween 80 and the use of a second,
smaller phenyl-Sepharose column. PAH from rat liver is generally
purified with an iron addition step, resulting in stoichiometric
amounts of iron and improved activity. The conditions used for iron
addition, which involve the addition of ferrous iron to oxygenated
buffers in the presence of reductant, are associated with the
generation of reactive oxygen species that might damage PAH. Inclusion
of enzymes known to scavenge reduced, reactive oxygen species improved
the specific activity of the protein purified with an iron addition
step from 5 to 8 units/mg. ()Whether this observed increase
in specific activity results from the degradation of peroxide or
superoxide, or by some other mechanism, is unknown. In general,
purifications including an iron addition step did not result in rPAH of
higher specific activity despite the observation of more iron per
subunit in treated protein. This has also been observed with partially
purified rat hepatic enzyme(42) . Omission of this step
altogether results in protein containing near stoichiometric levels of
iron (0.7 iron/subunit), with specific activity that depends directly
on iron content. This implies that there is very little protein
containing inactive iron. This fractional specific activity is
consistently 10.5 ± 0.5 units/mg per 1 catalytically competent
iron atom/subunit.
Figure 3:
Dissociation of rPAH tetramers into dimers
as the pH is increased, detected by small-zone analytical gel
filtration monitored at 280 nm. A Superdex 200 26/600 column was eluted
at 2.5 ml/min with 0.12 M phosphate at 5 °C, at pH 6.0,
6.4, 6.8, 7.2, 7.6, and 8.0 (ionic strengths ranged from 86 to 122
mohm cm
). A common stock of rPAH
was prepared by purifying 30 mg of rPAH over Superdex 200 at pH 6.0. A
conservative cut from the tetramer peak was saved for rechromatography,
excluding aggregate, oligomer, and dimer fractions. Each injection was
of 200 µg of rPAH diluted freshly 38-fold in the elution buffer
(
4 µM [subunit] at t = 0);
all elutions were done on a single day to avoid artifacts due to
freeze-thaw cycles. Chart-recorder traces were manually digitized using
Mathematica. The data presented have not been corrected for small
variations in the amounts of rPAH injected.
These conclusions are supported by data obtained from
dynamic light-scattering studies. The relatively narrow dynamic range
of this experiment limited studies to 5 mg/ml rPAH (0.1 mM subunits in 0.1 M phosphate buffer). At this
concentration, the Stokes radius observed for rPAH was 51
± 2 Å. The same values are obtained at pH 6.0, 8.0, and at
pH 8.0 with 0.5 M KCl present. Unfortunately, data from
rPAH
samples were unreliable, apparently because of
aggregation.
Figure 4:
Spectra of ferric rPAH and
iron-depleted (apo) rPAH
at 25 °C. The holoenzyme T/R
spectra were obtained in 50 mM MOPS, 0.3 M KCl (pH
7.3) at 25 °C. R state enzyme was generated from T state by
addition of L-Phe to 1 mM followed by a 5-min
incubation at 25 °C. The contribution from buffer and for the R
state spectra, L-Phe, has been subtracted. The apoprotein
sample was prepared by treating 2 mg of rPAH (specific activity, 6.0
units/mg, 0.9 iron/subunit) with 1 mMo-phenanthroline and 1 µl of 10%
-mercaptoethanol in a total volume of 250 µl. The increase at
510 nm due to the formation of
[Fe(o-phen)
]
was
monitored at 25 °C for 60 min, by which time
94% of the iron
had been removed. Small molecules were removed by passage over a column
(0.8
6.3 cm) of Sephadex G25 (medium grade, Pharmacia)
equilibrated in 0.1 M phosphate, 5% glycerol buffer (pH 6.8).
Glycerol is present to stabilize the apoprotein and facilitate gel
filtration, but it tends to exaggerate the molar absorptivity of PAH. A
control holoenzyme rPAH
sample that was exchanged into the
same 5% glycerol buffer shows a
15% more intense absorbance at 280
nm and stronger absorptions above 290 nm. Inset, difference
spectrum showing new chromophores formed upon incubation of ferric
rPAH
with catechol. 20 equivalents of catechol were added
to 1.06 mg of rPAH
(specific activity, 5.3 units/mg, 0.96
iron/subunit) in a final volume of 0.8 ml in 50 mM MOPS, 0.3 M KCl (pH 7.3) at 25 °C. Using
= 1900 M
cm
determined by active site titrations (described in text), 15
µM of the catechol-Fe
adduct forms
(subunit concentration = 25.7 µM), indicating that
the fraction of ``active'' iron is 0.58. Uncomplexed catechol
does not absorb above 400 nm under these conditions (data not shown),
and its contribution has not been subtracted from the spectrum.
Catechol stocks were standardized in methanol, using
= 2700 M
cm
(Sadtler Research Laboratories, Ultra Violet Spectra 108 UV,
Philadelphia).
The
fluorescence spectrum of PAH is dominated by the characteristic
emission of three tryptophans, which can be selectively excited at 293
nm (Fig. 5). The fluorescence emission of activated rPAH is
red-shifted relative to resting enzyme, which is also typical of rat
hepatic enzyme(52, 53) . In addition, the
rPAH fluorescence is more susceptible than that of
rPAH
to quenching by small molecules, whether anionic or
neutral. Quenching of the protein fluorescence by acrylamide or by
potassium iodide yielded Stern-Volmer plots analogous to those using
comparable concentrations of free tryptophan. This is interpreted as an
indication that the fluorophore exposed upon activation behaves as if
it were a single tryptophan moving closer to bulk solvent(53) .
These observations are also consistent with the observed increase in
hydrophobicity upon activation that is exploited in the purification
procedure.
Figure 5:
Fluorescence emission spectra at 25 °C
of 70 µg/ml ferric rPAH (1.4 µM subunits) in 0.1 M phosphate buffer, pH 6.8, activated
where indicated with 10 mML-Phe. Samples were
purified over Superdex 200 to remove small molecules. Spectra were
recorded with vigorous stirring and are corrected for dilution.
Excitation wavelength was at 293 nm with a 2-nm slit width, while
emission slit width was 4 nm. Also shown is the effect of quenching of
each by 0.2 M acrylamide. Top, Stern-Volmer plots of
quenching due to acrylamide (5 values between 0 and 0.2 M) or
potassium iodide (5 values between 0 and 0.4 M). The T state
is represented by a solid line and filled circles,
and the R state is represented by a dotted line and open
circles.
The CD spectra of T (Fe and
Fe
) and R (Fe
and
Fe
) states of rPAH (Fig. 6), obtained at 4
µM [subunit] from 400 to 195 nm, were found to
be quite similar (Table 2). These spectra were analyzed with a
standard basis set to estimate a linear combination of four structural
elements, assuming a mean helix length of 10 residues(54) .
There appears to be a slightly increased fraction of helix in the R
state samples compared to T state. Overall, significantly greater
helical content was predicted than has been reported elsewhere for PAH,
while the distribution of random coil predicted is quite small compared
to a previous report. We report data to lower wavelengths (195 nm versus 210 nm) critical to the assignment of secondary
structure, which might explain the discrepancy with earlier
results(55) .
Figure 6:
Circular dichroism spectra of
oxidized/reduced rPAH at 25 °C in 20 mM phosphate buffer, pH 6.8, with 0.1 M KCl, at 0.2 mg/ml
rPAH (4 µM subunits). Each of the T state samples was
prepared by 2 min, 25 °C incubation of 24 nmol rPAH with either 48
nmol 6-MPH
or the equivalent volume of buffer (12 µl)
in a total volume of 90 µl. Samples were made 0.1 M in
KCl, passed over 2 ml P-10 (Bio-Rad) equilibrated in the same buffer,
which was then used to elute protein free of pterin. R states were
generated from these samples by addition of L-Phe to 1
mM, followed by a 5-min incubation at 25 °C to activate
the enzyme. All spectra were obtained at 25 °C. Pathlength =
0.5 mm, scan rate = 0.5 nm/s, 5 scans averaged per spectrum.
Spectra were base-line corrected using the average value of
250-300 nm as zero.
Figure 7:
Catechol binding to active iron in 50
mM MOPS, 50 mM KCl (pH 6.8). Absorbance at 698 nm
plotted as a function of catechol titrated into a solution of rPAH at 3
mg/ml (60 µM subunits; specific activity, 6 units/mg; 35
µM active iron-containing subunits). Each point is the
result of a 5-min, 10 °C incubation of rPAH at the indicated
[catechol]. Titration data were fit to the expression given
under ``Experimental Procedures.'' Inset, binding of
11 µM catechol to 11 µM rPAH at
18 °C monitored at 698 nm in 50 mM MOPS, 0.3 M KCl (pH 7.3). The solid line is a simple first-order fit
to the data with k
= 0.11
s
. Final chromophore corresponds to 9.0
µM, 8.8 µM expected from specific activity of
7.8 units/mg (102%).
The visible spectra of the catechol
adducts of ferric rPAH and rPAH
are similar to
a variety of mononuclear iron complexes(56) . Data from model
systems of similar coordination geometries containing ligands of
varying Lewis basicities and charge indicate that the catecholate LMCT
transitions are sensitive to the ligand environment and undergo a
bathochromic shift as the net basicity of the ligands is decreased (56) . As more highly charged oxyanionic ligands (carboxylates)
interact strongly with the ferric center, the metal center's
intrinsic Lewis acidity decreases, destabilizing metal d-orbitals relative to the filled catecholate orbitals thereby
inducing a hypsochromic shift in the LMCT spectrum. Decreasing the
basicity of the ligands (by exchanging carboxylate oxygen for N donors)
would result in weaker metal-ligand interactions and a smaller
catecholate-metal energy gap, which would induce a bathochromic shift
in the LMCT spectrum. This effect can be quite dramatic with the low
and high energy catecholate LMCT bands ranging from 800 and 495 nm
(tris-(2-pyridylmethyl)amine) to 550 and 388 nm (N-(4,6-di-tert-butyl-2-hydroxybenzyl)-N-(carboxymethyl)glycine),
with energy shifts of approximately 5700 cm
and 5550
cm
, respectively. While these data suggest that
catecholate-to-Fe
LMCT transitions are sensitive
probes of iron coordination environments, we are currently unable to
satisfactorily deconvolute the overlapping contributions of ligand
environment from factors such as solvent accessibility to active site,
protein matrix environment (charge distribution, micro-dielectric
constant) near the iron site, and hydrogen bonding networks that are
known to induce significant shifts in the energy (1500-2000
cm
) of the LMCT bands for simple model complexes.
Figure 8:
Spectra A and B show EPR spectra
of ferric rPAH (specific activity, 6 units/mg, 0.9
iron/subunit) obtained in Tris (A) and MOPS (B)
buffers. In each case the buffer was 50 mM at pH 6.8 (5
°C). Spectrum C is the EPR spectrum of ferric rPAH
(specific activity, 6.6 units/mg, 0.67 iron/subunit) obtained in
5 mML-Phe, 50 mM MOPS, 0.3 M KCl
(pH 7.2). This sample was activated at 1 mg/ml by incubating at 25
°C for 10 min and then concentrated by ultrafiltration over a YM30
membrane to 16 mg/ml. Inset, The rPAH
lower
Kramers' doublet at g
= 8.8. For spectra A-C, scan time = 12.8 min; sweep width =
2000 G; modulation amplitude = 20 G; modulation power =
3.6 µW; temperature = 4.3-4.4 K. Spectra are not to
scale.
The electronic environment of
the ferric center of rPAH is changed by small coordinating
molecules such as Tris buffer. The axial EPR spectrum of rPAH in pH 6.8
Tris buffer (Fig. 8A) is significantly altered from the
spectrum in MOPS buffer with two new signals appearing at g
= 6.7 and 5.4, giving the EPR spectrum
previously reported for rat hepatic
PAH(34, 59, 60, 61) .
A
significant change in the EPR spectrum is observed upon activation of
ferric rPAH by L-Phe (Fig. 8C).
The broad features observed in the rPAH
spectrum are
replaced by a new, prominent resonance at g
= 4.5 whose intensity decreases with decreasing rPAH
specific activity, indicating that it is associated with the
catalytically competent iron of rPAH. Activation significantly
decreases the apparent micro-heterogeneity that is characteristic of
the rPAH
EPR spectrum, suggesting a tightening of the
distribution of iron environments under these conditions. The
electronic environment of the inactive iron at g
= 4.3 observed in both the rPAH
and rPAH
samples appears to be unaltered by the activation process. These
data are equivalent to those observed for the rat hepatic enzyme.
Non-activating exogenous ligands, in addition to Tris, sharpen
the EPR resonances of rPAH. The spectrum of the 1:1 adduct
of catechol and rPAH
(Fig. 9A) indicates a
complex with nearly axial environment with resonances at g
= 7.45 and 4.27 from the lower Kramers
doublet and g
= 5.82 from the middle
Kramers doublet, characteristic of an iron site with E/D
0.07. It is not clear whether the catechol binds as a mono-
or bidentate ligand, though resonance Raman studies were interpreted to
support bidentate ligation(62) . The spectrum resembles that
obtained for bovine adrenal TyrH, which copurifies with a tightly bound
catecholamine(63) .
Figure 9:
Spectrum A is the EPR spectrum of the 1:1
adduct of rPAH (specific activity, 6.0 units/mg, 0.66
iron/subunit) with catechol (20-fold excess) obtained in 50 mM MOPS, 0.3 M KCl (pH 7.2). Spectrum B is the
nitric oxide adduct of {Fe
} rPAH, which was
prepared in an argon box under anaerobic conditions.
6-MPH
-reduced rPAH (3.5 mg; specific activity, 8 units/mg;
0.7 iron/subunit) was diluted into 310 µl of degassed 100 mM MOPS, pH 7.3, with 50 mM KCl (three freeze-pump-thaw
cycles). Small aliquots of degassed 0.1 M sodium ascorbate and
1 M NaNO
were added alternately in three rounds to
a final volume of 1.4 ml. This was concentrated by centrifugation to
0.3 ml and loaded into the EPR tube, which was capped with a new
septum. Estimation of the E/D for
{FeNO}
from the indicated crossing points was
performed as described(93) . All EPR samples were frozen slowly
by suspension over liquid N
, since a suitable glassing
agent is unavailable for PAH(94) . Both spectra were recorded
with scan time 15 min; sweep width = 2000 G; modulation
amplitude = 20 G; modulation power = 1.8 µW;
temperature = 4.3-4.4 K. Spectra are not to
scale.
Ferrous PAH, which is the catalytically
relevant form of the enzyme, is more refractory to spectroscopic study
than the resting, ferric state of the enzyme. The modest
Fe-derived optical chromophores disappear, and
high-spin Fe
(S = 1) becomes
undetectable with standard EPR techniques, requiring specialized
methods (Mössbauer, MCD, EXAFS)(64) . The
binding of nitric oxide, however, generates a yellow, EPR-active
{FeNO}
adduct (S = ) with rPAH
that will allow its use as a sensitive probe of the geometry and
electronic properties of the ferrous active
site(65, 66) . The EPR spectrum of the nitric oxide
complex of ferrous rPAH
formed by exposure of the
pterin-reduced enzyme to ascorbate/nitrite under anaerobic conditions
is shown in Fig. 9B. This spectrum (g
= 4.12, g
= 3.88) is
essentially identical to those reported for the NO adducts of
isopenicillin N-synthase (g
=
4.09, g
= 3.95)(67) , the
non-heme iron site of photosystem II (g
=
4.09, g
= 3.95)(68) ,
protocatechuate 4,5-dioxygenase (g
=
4.09, g
= 3.91)(69) , catechol
2,3-dioxygenase (g
= 4.16, g
= 3.83)(37) , soybean
lipoxygenase(70) , and the model complex
[Fe(EDTA)(NO)]
(g
= 4.10, g
=
3.90)(71) . This signal originates from the ground state
Kramers' doublet (M
= ±
) that resides on a center with S
=
. The two doublets (M
= ±
,
± ) are split in energy by an amount
(
=
2D(1 + 3
)
) in the absence of an
external magnetic field. The parameter
(
= E/D) is a measure of the deviation from axial
symmetry of the environment of the axial spin where D and E are the axial and rhombic zero-field splitting parameters,
respectively. The value of
may be determined under conditions in
which g
H
D and kT from the
expression g
= g
[1 + (1 ± 3
)/(1 + 3
)
](37) , yielding for rPAH at pH
7.3
0.02. Double integration of this spectrum versus a standard of [Fe(EDTA)(NO)]
showed
>85% conversion of enzyme bound iron to the NO adduct (data not
shown). Furthermore, since the coordination of two NO molecules to the
ferrous center is expected to yield an integer spin system having
either no EPR spectrum or one significantly different from that shown
in Fig. 9B, it is likely that only one iron
coordination site is readily available for NO binding.
Figure 10:
Double-reciprocal data determined with
lysolecithin as activator, at saturating O and fixed
concentrations of 6-MPH
. Aliquots of rPAH
were
preincubated for 5 min in 0.5 mM lysolecithin (prepared
freshly as a 4 mM stock in water) before the addition of DTT, L-Phe, and 6-MPH
. L-Phe concentrations
used ranged from 0.02 to 5 mM, and 6-MPH
concentrations ranged from 25 to 150 µM. At high
[L-Phe], substrate inhibition occurred (see text). Inset, analogous determination with L-Phe as
activator. Each was incubated at the given concentration of L-Phe for 10 min at 25 °C, which is sufficient to activate
the enzyme fully. Initial velocities given are from assays that differ
from the standard assay only by variation of each substrate's
concentration.
To extend the
range of the double-reciprocal plot, we used PAH activated with 0.5
mM lysolecithin, a structurally dissimilar, non-substrate
activator. This allowed (Fig. 10) the determination of initial
velocities at constant [rPAH] over a wide range of L-Phe concentrations, limited only by the sensitivity of the
assay (useful between 5 and 35 nmol Tyr min ml
)(31) . The constants determined
from this double-reciprocal plot are quite similar to those arising
from the L-Phe-activated rPAH data, with the exception of K
, which is higher in the L-Phe-activated experiment. This provides further support that
under standard steady state conditions, either L-Phe or
lysolecithin activation results in a ``functionally
identical'' state of the enzyme(33) . A comparison of
these values is given in Table 3.
The
simulation of tyrosine buildup curves from standard (UV/visible spectra
and stopped-flow) assay data afforded the simultaneous determination of
both the catalytic (k) and inactivation (k
) rates (Fig. 11). The temperature
dependence of k
values from such simulations
agreed well with simple linear approximation of tyrosine buildup in the
first 30 s (UV/vis, 14.6 ± 0.6 kcal/mol subunit; stopped-flow,
14.6 ± 0.3 kcal/mol subunit). From simulated values of k
, we determined the kinetic barrier to
irreversible inactivation to be 10 ± 1 kcal/mol subunit with a t of 4.3 min at 25, ° C.
Figure 11:
Arrhenius activation parameters under
standard PAH assay conditions, analyzed by direct simulation using the
model described in Fig. 1. The temperature dependence of the
initial velocity of reduced, L-Phe-preincubated enzyme
corresponds to an activation energy for the catalytic process of 14.6
± 0.6 kcal (mol subunit). An identical result
was obtained from the stopped-flow assay method (14.6 ± 0.3 kcal
mol
). The enzyme inactivates during L-Phe
hydroxylation, with a half-life of 4.3 min at 25 °C. This process
has an apparent activation energy of 10 ± 1 kcal (mol
subunit)
. Standard assay data were obtained with 7
µg/ml rPAH (0.14 µM subunits) at a specific activity
of 8 units/mg. The stopped-flow data that are shown in the figure were
obtained with 5 µg/ml rPAH (0.1 µM subunits) at a
specific activity of 6 units/mg. The error bars for the
catalytic rate data fall within the
symbols.
The direct observation of the
complex activation step (k in Fig. 1) is
more difficult with rPAH than with rat hepatic PAH under identical
assay conditions. Using a stopped-flow assay and 6-MPH
as
cofactor, we were able to observe a significantly larger fraction of
fully activated velocity present in the unpreincubated rPAH compared to
authentic PAH. We were unable to find a value for k
by direct simulation with values of k
and k
, determined in parallel with L-Phe-preincubated enzyme, without resorting to the assumption
that a fraction of unpreincubated rPAH is already in an R state (or
determining a k
for rPAH
). The
reason for this discrepancy with PAH from different sources is unknown
and is currently under investigation.
We report the overexpression in E. coli and
characterization of recombinant PAH, which has kinetic and physical
properties very similar to enzyme isolated from rat liver (Table 3). The observation of tyrosine formation is only one of
several important functional aspects of this enzyme. Other reports of
PAH expression in vitro have appeared, but in neither of these
has a complete description of these essential elements of PAH's
mechanism been reported(22, 43) . Such data will be
required to understand individual mutations that cause PAH dysfunction
and thereby PKU. It is not obvious from an examination of the nature or
location of the PKU-associated mutations what is nonfunctional in PKU.
Their distributed nature suggests that much of the highly conserved
sequence of PAH is carefully balanced to achieve tight regulation of L-Phe catabolism and may be quite sensitive to even
``conservative'' amino acid substitutions. PAH's normal
kinetic complexity reflects the requirement for several configurational
changes in the enzyme, some of which may be disrupted or disfavored in
PKU. The activities of several PKU mutants have been estimated, several
with 10% residual activity(21) . While this can be
physiologically quite serious, it may only be caused by a difference of
a few kcal mol
in one of the activation barriers for
allosteric activation, catalysis, or some other essential feature of
PAH. To establish a solid linkage of phenotype with genotype, one needs
a detailed structural description of PAH and PAH variants.
As
isolated from rat liver, the specific activity of PAH varies from batch
to batch in a manner that is attributable to the fraction of
``active'' iron, as distinguished from sites containing
``inactive'' iron. Inactive iron cannot perform the
hydroxylation reaction, and efforts to restore it to an active state
have proven unsuccessful(3) . Inactivation of catalytically
competent iron sites is particularly rapid during catalysis; the
activity of PAH diminishes in a first-order manner (33) with a
half-life of 4.3 min at 25 °C. In addition to the uneven
phosphorylation of PAH, allelic heterogeneity is present in the common
strains of laboratory rats(73) . The fact that PAH forms a
tetrameric structure amplifies all of these sources of heterogeneity.
By heterologous expression of a single allele of rat hepatic PAH, we
avoid a number of these problems. There are no known post-translational
modifications of PAH (8, 46, 74) other than
phosphorylation (which is easily performed by purified protein kinase (75, 76) ), that would require expression of PAH in a
eukaryotic cell line. In addition, E. coli does not contain a
suitable pteridine cofactor(77) , which prevents induced rPAH
from performing hydroxylation, and thereby autoinactivating, prior to
purification. This results in our observation that nearly all of the
rPAH-associated iron is bound in the ``active''
configuration. The successful overexpression of this oligomeric,
mammalian protein in E. coli is in conflict with assertions to
the contrary (22) and in agreement with a similar
accomplishment by Kaufman and co-workers (43) . ()
Overexpression of active rPAH tetramers in E. coli confirms that correct quaternary assembly occurs. When activated,
rPAH can consume L-Phe at the same rate as rat liver PAH. The
recombinant enzyme also shows inhibition of PAH activity at low levels
of substrate; this sigmoidal response to L-Phe is indicative
of allosteric behavior. In vivo, L-Phe levels may
depend directly upon PAH allostery, since they are not depleted below a
threshold value equal to the steepest part of PAH's in vitro activation response(33) . Allosteric activation occurs in
the rPAH protein following incubation with either L-Phe or
lysolecithin, the most commonly used activators of the rat hepatic
enzyme. Activation is correlated with characteristic structural changes
in the rPAH enzyme as well as the expected increase in initial
activity. Light scattering and gel filtration experiments performed on
rPAH and rPAH
indicate that there is a
significant increase in the tetramer's size upon activation,
which is consistent with the Monod, Wyman, and Changeux (MWC) model for
an enzyme undergoing cooperative activation(78) . The observed
3.6% increase in the solution radius is typical of allosteric proteins
undergoing homotropic activation; E. coli aspartate
transcarbamoylase undergoes a 5.4% increase upon binding of N-(phosphonacetyl)-L-aspartate(79) , and
yeast phosphofructokinase undergoes a 4.3% increase upon ATP
binding(80) . Lysolecithin activation at equilibrium has a
small Hill coefficient, which is consistent with this structurally
distinct compound activating PAH by a different mechanism than occurs
during L-Phe activation. Regardless of the mechanism of activation, PAH
and
PAH
have essentially identical
catalytic properties, including expanded substrate specificity, and are
structurally similar, including a characteristic increase in
hydrophobicity(3) . In simply referring to
``activation'' or ``activated enzyme,'' the
mechanisms by which PAH becomes fully active are commingled with the
kinetic characteristics of that state. We prefer the use of the Monod,
Wyman, and Changeux nomenclature (T and R) to refer to the two
particular configurations of PAH that are associated with distinct
kinetic characteristics(78) , distinguishing formal allosteric
activation from generally stimulatory treatments. Some treatments that
increase enzyme activity appear to arise from genuine changes in enzyme
structure (high pH, phosphorylation, limited proteolysis), but others
seem uncorrelated with such changes (``spontaneous
activation''(4) ).
A comparison of the spectroscopic
properties of rPAH and rPAH
is presented in Table 2and Table 4. While the observed differences in
Stokes radii, fluorescence, and CD spectral data are consistent with an
overall protein structural change upon conversion of rPAH
to rPAH
, the fate of the coordination environment
about the iron center is less well defined. Although the energies of
the LMCT bands in the optical spectra of native rPAH
and
rPAH
as well as those of their respective catechol adducts
are equivalent, suggesting that the iron primary coordination sphere
remains relatively unchanged, the EPR spectra of these states clearly
indicate a change in the iron site electronic structure. Owing to the
similar affinities of the independent allosteric effector site (110
µM, pH 6.8) and the catalytic site (180 µM,
pH 6.8) for L-Phe, it is expected that both sites will be
fully occupied under the experimental conditions generally used for the
complete conversion of rPAH
to rPAH
. We are
therefore unable to fully assess whether the observed changes in the
EPR spectra are a consequence of the effect of substrate in the active
site or by changes in the iron coordination environment induced by the
structural reorganization accompanying the rPAH
to
rPAH
conversion. However, the electronic spectral data are
consistent only with the former possibility. A more extensive study of
the EPR and Mössbauer spectra of the various states
available to rPAH will appear elsewhere. (
)
The absence of
phosphate in the E. coli-expressed rPAH further confirms that
the low, variable levels of phosphate content in liver protein are not
required for, nor are they equivalent to, allosteric activation. The
half-maximal activation response of unphosphorylated rPAH occurs at the same concentration as partially phosphorylated
(0.2-0.3 phosphate per subunit) PAH. This suggests that the
stimulatory effect of phosphorylation is not expressed at low levels of
phosphate content. The observation of a K
identical to that of rat hepatic PAH confirms that
phosphorylation affects only the regulatory L-Phe site; it
does not perturb the active site L-Phe affinity or any
catalytic properties. Only L-Phe and lysolecithin have been
shown to cause a cooperative, sigmoidal activation response (at
[effector] < 1 mM), using either BH
or 6-MPH
. Phosphorylation sensitizes
the enzyme to its substrate by lowering the concentration of L-Phe necessary for activation (9) without affecting
the sigmoidal shape of the response, causing more effective in vivo catabolism(81) .
Hydroxylation of L-Phe by
rPAH requires molecular oxygen, L-Phe, and a pterin cofactor,
with maximal activity at one atom of iron per subunit. The remarkable
chemistry performed by PAH depends upon the correct positioning of
these substrates near the active site iron. This occurs with either
6-MPH or the natural cofactor BH
. Variations in
the structure or orientation of this side chain (or addition of a
7-substituent), which does not participate directly in the reaction,
can lead to ``uncoupling'' of the hydroxylation of L-Phe from the oxidation of tetrahydropterin(82) .
Both pterins are efficiently used as cofactors for rPAH, but the
initial time course of a standard assay using BH
has a
pronounced curvature (data not shown) that makes the assignment of
initial velocities difficult. Shiman and co-workers (11) have
recently reported similar but less pronounced phenomena in a study of
rat hepatic PAH, which they ascribed to BH
-dependent
relaxation of the activated enzyme (R
T)(11) . The
inhibitory characteristics of BH
are well known, among them
prevention of substrate activation (3) and inhibition of
phosphorylation(9) . Full phosphorylation of the enzyme may
obliterate the BH
regulatory site(9) . The
observations are consistent with BH
acting as a classic
negative allosteric effector, i.e. one that preferentially
binds to and stabilizes the T state. The determination of an Arrhenius
activation energy for rPAH's allosteric activation has been
hampered by a kinetic difference in the T
R conversion process
of rPAH. However, activation by L-Phe at equilibrium causes 12-fold stimulation of the initial velocity, occurs at the
same concentrations of applied activator, and yields a similar Hill
coefficient.
Prereduction of ferric PAH is required for activity and
can be detected as an initial absorbance decrease in a standard assay
of oxidized rPAH performed on a stopped-flow. Reduced
PAH
binds both of its substrates and a molecule of
O
, which is activated for hydroxylation within a complex of
pterin, L-Phe, and {Fe
} PAH. The
mechanism of O
activation and the identity of the
hydroxylating intermediate are unknown. Pterin does not appear to
coordinate directly to the active iron (83) but is generally
accepted to be an essential component of the active site. The catechol
and NO adducts of the ferric and ferrous forms of the enzyme will be
quite useful in assessing the catalytic importance of vacant iron
coordination sites, in the presence and absence of the tetrahydropterin
coenzyme.
Earlier studies designed to examine the steady state
equilibrium kinetic mechanism of PAH were apparently performed prior to
the realization that thorough preactivation of PAH is required for
maximal enzyme activity. The determination of a sequential mechanism
for PAH indicates that all three substrates are ordered at the active
site before any product is released. Reliance upon L-Phe
activation under steady state conditions severely limits the range of
concentrations available for study(72) . The L-Phe
activated results alone do not allow the assignment of a sequential or
ping-pong mechanism since parallel and intersecting lines are difficult
to distinguish over a narrow range of substrate
concentrations(84) . Lysolecithin allows the assay of rPAH at
low concentrations of [L-Phe] without complications
due to the activator role of this substrate. Lysolecithin was
maintained at 0.5 mM throughout the modified standard
assay to ensure stable activation of rPAH. In this way, the
double-reciprocal analysis of PAH at saturating O
has been
extended across more than 3 orders of magnitude of
[L-Phe] centered upon its K
.
This study confirms that a sequential mechanism is operative in rPAH
under conditions of lysolecithin activation, where the L-Phe-activated results are ambiguous at best. At high levels
of L-Phe and at [6-MPH
]
0.5
K
In summary, we have compared the kinetic requirements of rPAH to rat hepatic PAH in detail and find that the recombinant enzyme recapitulates every catalytic detail. There is stimulation of the rate of hydroxylation following exposure to the same allosteric activators; rPAH has the same requirement for reduced iron and the same affinity for its substrates. The temperature dependence of the catalytic step of rPAH yields a similar kinetic activation energy barrier. In addition, we have measured an Arrhenius activation barrier for the poorly understood kinetic inactivation process of PAH. Overexpressed rPAH is synthesized quickly (in 6 h versus several days for a eukaryotic expression system(22) ) in an environment free of pterin cofactor and general phosphorylating conditions, which results in a homogeneous preparation free of complications due to inactive iron and uneven phosphorylation. By many structural criteria, rPAH is essentially equivalent to rat hepatic PAH.
The availability of this source of rPAH will aid in the resolution
of several long-standing issues pertaining to the chemistry and
structure of PAH. One of the most important is the nature of the
allosteric activation process (T R conversion), which we have
shown by UV-visible spectra and CD spectroscopy causes only minor
changes in the carboxylate/histidine coordination environment of the
active site iron and the overall secondary structure of the protein.
There is at least one partially accessible, labile coordination site on
the iron (Fe
and Fe
) present in T
state rPAH, demonstrated by the ready formation of Tris, catechol, and
NO adducts. However, rPAH
is able to preclude access to the
iron site by its substrates, directing them instead to the allosteric L-Phe site and the pterin reduction site, which are
functionally and/or spatially distinct from the rPAH
active
site. In support of this, EPR spectra show that both T and R state PAH
have mostly rhombic active site environments, but in the latter case
increased L-Phe accessibility to the active site causes
perturbations due to its binding near the iron.
Spectroscopic data are consistent with the immediate iron environment
being relatively insensitive to the allosteric state of the enzyme.
Characteristic fluorescence shifts, increased hydrophobicity, a large
activation energy barrier, and a 10% volume increase are suggestive of
a large reconfiguration of the distant protein matrix following
allosteric activation. This process can now be more reliably envisioned
as resulting from the removal of an inhibitory portion of the protein,
exposing a functional active site, rather than as a L-Phe-dependent rearrangement of the iron ligands.