(Received for publication, June 13, 1995; and in revised form, September 27, 1995)
From the
Mass spectrometry has been used to demonstrate that vitamin
K-dependent carboxylation is a processive post-translational
modification (i.e. multiple carboxylations occur during a
single association between enzyme and substrate). Purified vitamin
K-dependent carboxylase can carboxylate as many as 12 glutamate
residues in FIXQ/S, a peptide substrate based on amino acids -18
to 41 of the human blood clotting enzyme factor IX. Mass spectrometry
was used to determine the number of -carboxyl groups added to
FIXQ/S by the carboxylase during an in vitro reaction. Despite
the fact that most substrate molecules in a reaction were
uncarboxylated, almost all carboxylated FIXQ/S molecules were
carboxylated many times. This observation can only be explained by two
types of mechanisms. In a processive mechanism, multiple carboxylations
could occur during a single substrate binding event. Alternatively, a
distributive mechanism could result in the observed behavior if the
initial carboxylation event results in a substrate that is additionally
carboxylated far more efficiently than the uncarboxylated FIXQ/S.
Kinetic experiments and arguments were used to show that the vitamin
K-dependent carboxylase is not distributive but rather is one of the
first well documented examples of an enzyme that catalyzes a processive
post-translational modification.
The vitamin K-dependent carboxylase converts glutamate to
carboxyglutamate (Gla) by the addition of CO at the
-position(1, 2, 3, 4, 5) .
In addition to glutamate and CO
, the reaction requires the
cosubstrates O
and reduced vitamin K. The native substrates
for the carboxylase include several proteins central to blood clotting
as well as bone Gla protein, matrix Gla protein, and others. Each of
these proteins contain a ``Gla domain'' in which glutamate is
converted to Gla. This Gla domain binds Ca
and is
essential for activity in all of the identified Gla-containing
proteins.
Efficient carboxylation of the native substrates requires
that they contain a conserved region which binds to the carboxylase
with a submicromolar affinity constant(6, 7) . In the
blood-clotting factors, this region is contained in an amino-terminal
propeptide which is proteolytically removed following carboxylation.
Substrates containing this propeptide have K values 3 to 4 orders of magnitude lower than small peptide
substrates that do not contain the
propeptide(8, 9, 10) . This suggests that the
propeptide acts primarily as a ``docking'' site which
localizes the glutamate residues near the carboxylase active site.
Using the vitamin K-dependent carboxylase activity present in
solubilized bovine microsomes, we previously reported the introduction
of multiple Gla residues into peptide substrates derived from the
propeptide and Gla domain of human Factor IX(9) . In this
study, we use one of those substrates, FIXQ/S (Fig. 1), to
investigate the mechanism by which multiple glutamates per substrate
are converted to Gla residues during vitamin K-dependent carboxylation.
As a consequence of our recent purification of the vitamin K-dependent
bovine carboxylase(11) , we can perform in vitro carboxylations from which the carboxylated products can easily be
recovered. After suitable purification, the number of Gla residues
introduced into the peptide products can be determined by mass
spectrometry on the products as they elute from a reverse phase HPLC ()column. We also found that elution of the various
carboxylation states from an anion exchange HPLC column is closely
correlated with the extent of carboxylation. This behavior allowed us
to perform a series of kinetic experiments to demonstrate that the
vitamin K-dependent carboxylase is truly processive.
Figure 1:
Sequence of the human FIX-based
substrate FIXQ/S. The propeptide and Gla domains are separated by the
tilde () symbol. The 12 glutamates that are converted to Gla in
the native Factor IX are in bold type. Although these changes
are unlikely to affect carboxylation, residues that have been mutated
from the native Factor IX are underlined.
Examples of
processive enzymes are very common in the replication and modification
of RNA and DNA(12) . However, examples of processive
post-translational modification for which there is good supporting
evidence appear to be rare. Nevertheless, processive modes of
post-translational modification should offer significant advantages in
a wide variety of biochemical processes where multiple identical
modifications are introduced into a substrate. The few apparent cases
of such modifications in the scientific literature reflect this
diversity and suggest that processive post-translational modification
may be very common. Unlike processivity involving nucleic acids,
processive post-translational modification of proteins may often occur
in patterns other than a linear sequence along the polymer chain. The
vitamin K-dependent -glutamyl carboxylase appears to be an
excellent model for studying such processes.
The peptide substrate FIXQ/S was expressed in Escherichia coli as a fusion protein and cleaved with cyanogen bromide as described previously(9) . The peptide products dissolved in guanidine HCl were dialyzed against 25 mM MOPS (pH 8.0) with 50 mM NaCl. The soluble FIXQ/S product was collected as described previously(9) . Formylation of cysteine was reversed by incubating FIXQ/S with 20 mM DTT for 4 h at 25 °C(14) . The peptide-containing solution was then chilled to 4 °C and loaded onto a DEAE-Sepharose column (20 ml of matrix per 50 mg of protein) equilibrated with 25 mM MOPS (pH 8.0) and 50 mM NaCl. The column was washed with 2 volumes of equilibration buffer, and the protein eluted in 25 mM MOPS (pH 8.0) with a NaCl gradient from 50 to 500 mM NaCl.
Vitamin
K (Abbott Laboratories) at 10 mg/ml was reduced by addition
to 3 volumes of buffer containing 200 mM DTT, 500 mM NaCl, and 20 mM Tris (pH 8.5). Full tubes were incubated
in darkness at 4 °C for at least 36 h to ensure complete reduction.
To perform a
reaction, most of the solution components were combined and placed on
ice. After the solution had chilled, purified carboxylase which
represented 20% of the final reaction volume was thawed and added to
the ice-cold solution. The NaHCO
was then
added, and the solution was placed in a 17 °C water bath for a 1-h
preincubation. The reaction was initiated by addition of vitamin
KH
and allowed to proceed for as long as 44 h at 17 °C.
The progress of the reaction was followed by periodically removing
small samples and assaying them for incorporated
NaHCO
as in previous FLEEL
assays(15) . At the desired time, large reactions were
terminated in a fume hood by the addition of acetic acid to a final
concentration of 30 mM, resulting in a pH below 5. The
remaining steps were done at room temperature in a fume hood. The
NaH
CO
was removed by bubbling N
gas through the reaction solution for about 30 min. The
CO
gas was collected by running the exhaust
gas through two consecutive traps containing 0.5 N NaOH. The
reaction container was sealed except for tubes leading from the
nitrogen and to the NaOH trap. The tube from the nitrogen went to the
bottom of the reaction vessel and could be used to remove small amounts
of solution. After scintillation counting revealed that more than 99.9%
of the free radioactivity had been removed, reaction solutions were
neutralized by addition of NaOH to a final concentration of 30 mM and diluted with 1.5 volumes of deionized water. The diluted
reaction was combined with 0.04 volume of a 50% slurry of
DEAE-Sepharose in 10 mM Tris, pH 7.3, and 50 mM NaCl.
The matrix was recovered in minimal volume and loaded into a 2.5-cm
diameter column. The column was washed with 2 volumes of column
equilibration buffer and eluted with 10 mM Tris (pH 7.3) and 1 M NaCl. Fractions containing
C-FIXQ/S were
determined by scintillation counting a portion of each sample. These
fractions were then pooled and frozen. In all experiments and at all
stages, peptide samples were frozen in liquid nitrogen and stored at
-70 °C. All fractions were collected and stored in
polypropylene Microfuge tubes.
To remove uncarboxylated FIXQ/S, fractions containing labeled FIXQ/S were concentrated, desalted, and fractionated a second time on the Hydropore AX column as described above. Eluted fractions were then pooled into groups and frozen. During the above procedures, protein was quantitated from peak area relative to known amounts of FIXQ/S using absorbance at 220 nm.
After
thawing, each group was centrifuged to remove particulates and loaded
directly onto a 0.46 50 mm Rainin C18 column (83-203-F5) with
matching guard column. Samples were loaded using multiple injections
over 10 min during which buffer A (0.1% trifluoroacetic acid) was
flowing at 1 ml/min. After the load period, the flow rate was linearly
reduced over 1 min to 0.125 ml/min followed by 2 min at this velocity.
Next, a linear elution gradient brought the mobile phase composition to
100% B (0.1% trifluoroacetic acid, 90% acetonitrile) over 10 min. The
remaining 15 min of each run was 100% B. Protein-containing fractions
were collected, frozen in liquid nitrogen, and dried by rotary
evaporation. Protein was quantitated from peak area relative to known
amounts of FIXQ/S using absorbance at either 220 or 280 nm. Each group
was resuspended to 1 µg/µl in 25 mM ammonium hydroxide
after which acetic acid was added to a final concentration of 15
mM. Each group was aliquoted and frozen.
Mass measurements were made on a Fisions-VG Quattro BQ triple quadropole mass spectrometer equipped with a pneumatically assisted electrostatic ion source operating at atmospheric pressure. Mass spectra were acquired in the continuum mode from m/e 700-1400 with a scan time of 10 s. The mass scale was calibrated with horse heart myoglobin (16951.48 kDa) with a resolution corresponding to a peak width at half-height of 1.4 Da for m/e 893. The mass spectra were transformed to a molecular mass scale using software supplied by the manufacturer.
The average carboxylation state of carboxylated products was estimated using the assumption that the ionization efficiencies of all carboxylation states within a group were the same. From the relative amounts of each carboxylation state in the mass spectrometry profile for a group, the average carboxylation state of the carboxylated products in the group was estimated. The average carboxylation state for each group and the amount of carboxylated peptide in each group was then used to determine the overall average carboxylation state of the product peptides.
The carboxylation state of the reaction products from these experiments was estimated from their elution position on the Hydropore AX anion exchange column. For these experiments, the preparative anion exchange HPLC method was modified to the extent that elution gradients were linear from 0-100% buffer B and occurred over 25 min at a flow rate of 1 ml/min.
Figure 2:
Time course of carboxylation of FIXQ/S is
linear following an initial burst of activity. The 350-ml reaction
shown included 1,300 nM FIXQ/S and 20 nM carboxylase.
Projecting the linear portion of the time course to the axis suggests
an apparent burst size of 350 nM. Mass spectrometry data
presented below indicates that the specific activity of the
incorporated NaHCO
was 40.3 mCi/mmol. This
value was used to calculate the incorporated
CO
concentration. After 27 h of incubation, both the carboxylated
and uncarboxylated FIXQ/S from this experiment were recovered as
described under ``Materials and
Methods.''
In
enzymatic reactions, a rapid burst of product formation followed by a
slower linear rate of reaction often indicates that product release is
rate-limiting for the reaction. These burst phenomena are a consequence
of the accumulation of product-enzyme complex from a single substrate
binding event and catalysis. In this situation, the size of a burst may
indicate the concentration of active enzyme in the reaction and can be
determined by linear extrapolation of the linear portion of the
progress curve back to zero time. For the experiment in Fig. 2,
this method suggests a burst size of 350 nM incorporated
CO which is well above the 20 nM carboxylase
concentration estimated to be present in the reaction. However, it is
reasonably close to the concentration of CO
one would
expect to incorporate into 20 nM FIXQ/S if an average of about
7.5 CO
molecules are incorporated per FIXQ/S molecule (e.g. 150 nM). Data presented later suggest that this
average is approximately correct. Imprecision in the quantitation of
the carboxylase, which was done on SDS gels by comparison with IgG,
could easily account for the remaining difference. The presence of a
burst phenomenon which could be explained by multiple carboxylations
per binding event suggests a processive reaction with a rate-limiting
product release step.
Figure 3: Anion exchange HPLC on post-reaction FIXQ/S separates the carboxylated and uncarboxylated forms. A small portion (0.5%) of the FIXQ/S from the reaction in Fig. 2was loaded on a Rainin Hydropore HA weak anion exchange column and eluted with a gradient of the composition indicated. Buffer A contained 20 mM Tris, pH 7.2, while buffer B additionally included 2 M NaCl. For more detail, see ``Materials and Methods.''
Figure 4: Consecutive anion exchange column runs reduce the amount of uncarboxylated FIXQ/S that coelutes with carboxylated forms of FIXQ/S. FIXQ/S from the reaction in Fig. 2was desalted and loaded onto the Rainin Hydropore HA anion exchange column (data not shown). Carboxylated FIXQ/S fractions containing fractions from the first anion exchange elution (as indicated by the presence of radioactivity) were combined, desalted, and rerun as before. The FIXQ/S from the second elution (shown above) was pooled into six groups labeled G1 to G6 as indicated. Quantitation during reverse phase HPLC indicate that the groups contained the following amounts of protein: G2, 44 µg; G3, 33 µg; G4, 20 µg; G5, 40 µg; G6, 143 µg. See ``Materials and Methods'' for details.
Since increasing carboxylation of a peptide would result in a more negatively charged product, it seemed reasonable that more highly carboxylated peptides would elute from the anion exchange column at higher salt concentrations. As shown below, this behavior was observed and explains the broad range of salt concentrations at which the products elute from the anion exchange column.
Our initial attempts at mass spectrometry did reveal that the FIXQ/S starting material included multiple 28-dalton additions. This modification almost certainly represents formyl groups added to the cysteine residues during cyanogen bromide cleavage of the fusion protein and could be reversed by longer treatment with the reducing agent DTT(14) . It should be noted that serine or threonine residues may also be formylated by cyanogen bromide cleavage in formic acid; however, this modification should not be reversible with DTT(16) . A second species of peptide that was 28 daltons less than the observed mass of FIXQ/S was also observed. We have not been able to identify the modification responsible but it appears not to affect carboxylation (see below).
As a solution to
the problems mentioned above, we turned to direct mass spectrometry of
FIXQ/S as it eluted from a reverse phase microbore column. With this
technique, even groups containing highly carboxylated FIXQ/S yielded
high resolution data (Fig. 5). As indicated by their presence in
the later groups, the higher carboxylation states eluted from the anion
exchange HPLC column at higher salt concentrations. All 13 of the
possible carboxylation states (0 to 12 added CO groups) are
clearly evident. Each carboxylation state has a companion peak which is
28 daltons lighter than itself. As mentioned above, this modification
is evident in uncarboxylated FIXQ/S. The even distribution of this form
across the carboxylation states suggests that the modification has no
effect upon carboxylation. The observed molecular mass of
uncarboxylated FIXQ/S was 6954.2 daltons, in good agreement with the
expected average mass of 6954.6 daltons. The mass difference between
the carboxylation states is 45.3 daltons, corresponding to a specific
activity of 40.3 mCi/mmol for the incorporated carbon atom. The added
NaH
CO
had a specific activity of 56.4
mCi/mmol, but the presence of cold endogenous bicarbonate in the
reaction solutions results in some dilution.
Figure 5: Mass spectroscopic analysis of the groups 1 to 6 clearly shows FIXQ/S at every carboxylation state. The numbers near each peak indicate how many Gla residues would have to be present to yield FIXQ/S molecules of the observed molecular mass. The latter groups which eluted from the anion exchange column at higher salt concentration clearly contain more highly carboxylated FIXQ/S molecules. Prior to mass spectrometry, each group was desalted and purified by reverse phase HPLC. A portion of these purified samples were then loaded onto a C4 reverse phase microbore column and eluted directly into VG Bio Q mass spectrometer to obtain the above profiles. See ``Materials and Methods'' for details.
Quantitation of the average carboxylation state of the product peptides was simplified by the similar ionization efficiencies observed for the various carboxylation states. Under identical mass spectrometry conditions, the relative signal intensities from identical amounts of groups 2 though 6 in Fig. 5were, respectively, 96:100:80:66:51. The fact that the different groups have similar ionization efficiencies argues strongly that the ionization efficiencies of the different carboxylation states within a single group will have similar ionization efficiencies. This allows a reasonably accurate estimate of average carboxylation state of the products by the algorithm given under ``Materials and Methods.'' Because the peak containing highly carboxylated product (G6 in Fig. 5) contains most of the product peptide (Fig. 4), this material dominates the average, which is approximately 7.5 carboxylations per carboxylated product. While an estimate of the possible error in this average is difficult, even unreasonably extreme assumptions about relative ionization efficiencies of similar species within a group would result in a change in the average of less than 1 carboxylation per peptide.
Figure 6:
The
possible alternative mechanisms that could explain apparent
processivity during carboxylation. The carboxylation state of a product
is represented by the subscript (e.g. product
P is singly carboxylated). In the processive mechanism (A), released products do not reassociate with the
carboxylase. In the efficiency-enhanced distributive mechanism (B), binding and release are rapid. In addition, the
carboxylation of the first glutamate must be much less efficient than
additional carboxylations. Although it is not a necessary feature of
either mechanism, the conversion of products to a lower carboxylation
state (i.e. the back reaction) has been omitted because it is
energetically unlikely and has not been
observed.
Figure 7:
In
direct competition experiments, FIXQ/S with 1 or 2 carboxylations is
not a significantly better substrate than FIXQ/S. A 5-ml carboxylation
reaction was performed using C-FIXQ/S from group 3 (2.8
µg), 1 µM FIXQ/S (34.6 µg), 12 nM carboxylase, 0.38 mM cold NaHCO
. Propeptide
(proFIX19) was present in the reaction at 0.4 µM. The
reaction was initiated by the addition of 111 µM vitamin
KH
, supplemented with an additional 111 µM vitamin KH
at 12 h and stopped after 24 h. Products
were recovered and separated by anion exchange HPLC (same gradient as Fig. 8). A shows the radioactive profile of
C-FIXQ/S from group 3 before (
) and after (
)
reaction. Following the reaction, an additional 2.6% of the total label
shifted from the elution position of group 3 (1 or 2 carboxylations) to
the elution position of the Highly Carboxylated FIXQ/S (6 to
12 carboxylations). B shows the absorbance profile of the
postreaction FIXQ/S (upper profile) compared with prereaction
peptide (lower profile). The area under the peak labeled Highly Carboxylated FIXQ/S (shown enlarged as an inset) was estimated to be 1.2% of the total peptide
absorbance.
Figure 8:
Vastly lower concentrations of
carboxylated product do not affect the distribution of product
carboxylation states. A 200-ml reaction containing 1300 µM FIXQ/S and 0.1 nM carboxylase (200-fold below the
carboxylase concentration used in the reaction in Fig. 2) was
incubated for 1 h, resulting in the incorporation of 0.4 nMCO
into FIXQ/S. Despite the fact that at most
0.4 nM FIXQ/S molecules (1 in 3250) were modified, almost all
the incorporated radioactivity is found in molecules eluting at the
peak that contains product with 6 to 12 carboxylations. Note the
absence of an observable product peak in the absorbance profile
reflecting the low level of product formation. To dilute the
carboxylase without changing the reaction conditions, a solution
similar in composition to the buffer containing the purified
carboxylase was made (20 mM MOPS, 500 mM NaCl, 20%
glycerol, 2 mM EDTA, 0.1% phosphatidylcholine, and 0.7%
CHAPS). This was added to the reaction mixture prior to the carboxylase
so that the volume of the replacement solution plus the carboxylase
continued to be 20% of the total volume. The reaction contained 111
µM vitamin KH
and 4 nM proFIX19.
To
demonstrate the extent of this inconsistency, we performed a
carboxylation reaction with 1.3 µM FIXQ/S and a
carboxylase concentration of 0.1 nM. Under these conditions,
less than 0.4 nM NaHCO
was
incorporated into the peptide during the course of the reaction;
nevertheless, the extent of carboxylation as indicated by the elution
pattern of the radiolabeled peptide on anion exchange HPLC (Fig. 8) was essentially the same as observed previously (Fig. 3). For the vastly more abundant uncarboxylated FIXQ/S
(1300 nM) to be out competed by the carboxylated forms (below
0.4 nM) for an average of about 7.5 carboxylations would
require these forms to be carboxylated more than 3250-fold more
efficiently than FIXQ/S. As shown above, this was not the case. In
addition, over the range of enzyme concentrations which have been tried
(0.1 to 20 nM), the production of the carboxylated forms was
proportional to the carboxylase concentration (data not shown). Because
the concentration of enzyme affects the concentration of intermediates,
this would not in general be the case for an efficiency-enhanced
distributive mechanism.
On the other hand, both a similar distribution of carboxylation states and reaction rate proportionality would be expected from a processive enzymatic reaction. The concentration of carboxylase would not affect the distribution of carboxylation states resulting from an association between an enzyme and substrate. In addition, since the reaction velocity expected from a processive mechanism is not affected by the concentration of carboxylated intermediates, the processive mechanism would predict a carboxylation rate proportional to the enzyme concentration when the substrate concentration is essentially constant and not limiting.
A possible difficulty encountered in the above experiments was the presence of propeptide (FIX19) from the purified carboxylase that was used. Fortunately, the presence of propeptide should not affect the experimental outcomes. The concentration of FIXQ/S (1.0 µM) in these experiments was sufficiently above the concentration at half-maximal velocity (0.6 µM), observed in the presence of 0.4 µM propeptide (as in Fig. 7) that most of the carboxylase was bound to FIXQ/S. This observation shows that the carboxylated forms of FIXQ/S are competing primarily with uncarboxylated FIXQ/S rather than with propeptide.
Importantly, the relative reaction velocity is dependent upon the concentrations of both substrates. Extending this simple example to the more complicated situation in our carboxylation reaction, it is still clear that, as the concentration of the carboxylated intermediates increase, the relative rates of carboxylation of uncarboxylated and carboxylated FIXQ/S molecules must change, since the concentration of uncarboxylated FIXQ/S is essentially constant. Similarly, the relative rates of carboxylation of different carboxylation states should change relative to one another.
However, as data in Table 1show, during a time course of carboxylation from 1-22 h, the proportion of carboxylated peptide with about the same anion exchange elution times as Group 3/4 (1-3 carboxylations), Group 5 (3-6 carboxylations), and Group 6 (6-12 carboxylations) were unchanged. Thus, at changing levels of intermediates, additional uncarboxylated FIXQ/S continues to be recruited for carboxylation at the same rate as the carboxylated forms. Once again, this behavior is not consistent with an efficiency-enhanced distributive mechanism; however, it is exactly that expected from a processive mechanism as the concentration of unbound intermediates should have no effect on the distribution of carboxylation states resulting from an association between an enzyme and substrate.
For the in vitro carboxylation reaction, only two types of mechanisms could explain how as many as 12 carboxylations occur in carboxylated FIXQ/S molecules, while most substrate molecules remain uncarboxylated. One of these mechanisms, which we have termed the efficiency-enhanced distributive mechanism, is not consistent with the behavior observed for the vitamin K-dependent carboxylase. Neither was partially carboxylated FIXQ/S more efficiently carboxylated than FIXQ/S nor was the reaction velocity and the distribution of the carboxylation states dependent upon the concentration of partially carboxylated FIXQ/S as would be expected for this mechanism.
In contrast, the observed behavior of the carboxylase agrees with that expected from a processive mechanism in which multiple modifications of the substrate molecule occur during a single association between the enzyme and the substrate. In a processive mechanism, the degree of substrate modification should be unaffected by enzyme, substrate, or low partially modified product concentrations, since these variables should not affect the modification process once binding has occurred. In addition, the reaction velocity should be unaffected by low levels of partially modified intermediates and should be linearly proportional to the carboxylase concentration when substrate is not limiting. All of these behaviors were observed during the multiple carboxylation of FIXQ/S by the vitamin K-dependent carboxylase.
A processive carboxylation reaction is also consistent with the size and duration of the burst phenomenon observed during time courses of carboxylation. Given the inaccuracy of the method used to quantitate the carboxylase, the size of the burst is in reasonable agreement with the carboxylase concentration when it is recognized that an average of about 7.5 carboxylations occur during a single association between carboxylase and FIXQ/S. For enzymatic reactions where it has been demonstrated that burst behavior is a reflection of rate-limiting product release, the size of the burst is often the preferred method of determining the concentration of active enzyme. For the carboxylase, this suggests that gel quantitation results in an underestimation of the true carboxylase concentration by a factor of about 2.3-fold.
The processive carboxylation of an average of about 7.5 glutamate residues during a single enzyme/substrate association when combined with an average time per carboxylation per enzyme of 17 min implies that an average substrate molecule is remaining bound to the carboxylase for about 127 min. Thus, the half-time for a substrate to be bound is about 63.5 min, which qualitatively agrees with the time at which the burst is half-completed (see Fig. 2).
For each of the experiments presented, it may be possible to suggest unlikely formal possibilities that could result in the observed behavior. However, taken in total, we believe these experiments demonstrate that the vitamin K-dependent carboxylase is a processive post-translational modification enzyme.
It should be recognized that the partial carboxylation observed in
many of the in vitro reaction products is probably due to the
unnatural environment of the carboxylase rather than a characteristic
of the in vivo mechanism. It is difficult to imagine why
processive carboxylation in vivo would not proceed to complete
carboxylation of the substrate as complete carboxylation is necessary
for the expression of the calcium-dependent, functionally essential
domain conformation. Unanswered questions relating to the manner in
which the carboxylase recognizes complete carboxylation of its native
substrates are central to a full understanding of the mechanism of the
carboxylase. This is particularly true as carboxylation does not appear
to proceed in a linear sequence along the polymer chain, ()making this reaction very different from the linear
processivity observed in replication, transcription, and
translation(12) .
Interestingly, very few examples of the processive post-translational modification of amino acids have been reported. Most post-translational modifications for which the mechanism is understood involve binding to a particular amino acid and/or recognition site, performing a modification on a single amino acid and releasing the product. There are, however, examples of post-translational modification which are probably processive. Convincing evidence that ubiquitin-protein ligase (18, 19) is processive has been presented. Pulse/chase experiments with analysis during the presteady state period were used to demonstrate multiple ubiquitin additions during a single enzyme/substrate association. Evidence suggests that ubiquitination of histones by ubiquitin carrier protein (19, 20) and phosphorylation of RNA polymerase II by template-associated protein kinase (21, 22) may be processive. These three cases and vitamin K-dependent carboxylation are examples of a type of processivity that results from the association of enzyme and substrate at a binding site distinct from the modifiable residues, followed by modification at these residues.
A second class of processive
post-translational modifications may be represented by the
hydroxylation of proline by prolyl hydrolase(23) . Prolyl
hydrolase has two active sites and modifies protein collagen at a large
number of sites. The enzyme probably remains bound to at least one and
often two prolyl sites on the substrate for a period of time during
which many hydroxylations occur. Coincidentally, other work in our
laboratory may fit this method of processivity. The coagulation
protease Factor XIa exists as a dimer with two active sites. In the
intrinsic blood coagulation pathway, Factor XIa cleaves its substrate
Factor IX at two sites generating the active protease Factor IXa. In
our laboratory, both cleavages occur without the generation of an
intermediate. ()An apparent lack of intermediate products is
compatible only with a processive mechanism or with the less likely
efficiency-enhanced distributive mechanism.
Because processivity is possible for any enzyme which multiply modifies a substrate and which has two binding sites on the substrate, it seems likely that processive post-translational modification is a common phenomenon. Unlike processivity involving nucleic acids, processive post-translational modification will often occur in patterns other than a linear sequence along the polymer chain. This should have profound consequences for the mechanisms of processive post-translational modification enzymes. It appears that the mechanism of vitamin K-dependent carboxylation is an excellent model for studying these enzymes.