(Received for publication, August 1, 1995; and in revised form, September 27, 1995)
From the
The burial of charged residues in proteins is rare as it is
thermodynamically strongly disfavored. However, in ``standard
mechanism'' protein inhibitors of serine proteinases, the P residue, which is highly exposed, becomes buried in the S
specificity pocket of the enzyme. In many enzymes, such as Streptomyces griseus proteinase B (SGPB) the S
pocket is hydrophobic. We measured the pH dependence of the
association equilibrium constant for the interaction of SGPB with
turkey ovomucoid third domain P
mutants, Glu
OMTKY3 and His
OMTKY3. In order to eliminate the
effects of other ionizable groups on the enzyme and the inhibitor, we
divided these pH dependences by the pH dependence of the association
equilibrium constant for the Gln
OMTKY3 mutant. This
yielded for Glu
, pK
(free
inhibitor) of 4.46 ± 0.05 and pK
(complex) of 8.74 ± 0.06. For His
the
values are pK
6.63 ± 0.08 and
pK
4.31 ± 0.07. At low pH values
Glu
variant is a relatively good inhibitor for SGPB. This
may be biologically relevant.
Transferring a charge from a high dielectric medium, such as
water, to a low dielectric medium, such as organic solvents or
interiors of proteins, is not thermodynamically favored(1) .
Therefore, ionizable side chains buried in proteins should experience
large pK shifts. Such shifts are occasionally
seen(2, 3, 4, 5) , but they are
rare. While ionizable residues are fairly often buried in their
uncharged form, acquisition of charge is most often associated with a
conformational change (3, 4) or with dissociation of
the ligand-receptor complex. We describe two cases where the complexes
persist with the charged form of a buried side chain. Many more such
cases can be generated by examining the comparative pH dependence of
the association of ionizable P mutants of protein
inhibitors of serine proteinases.
The association of ``standard
mechanism'' (6) canonical (7) protein inhibitors
of serine proteinases is the most studied and best understood of
protein-protein interactions. In these inhibitors, each inhibitory
domain has a single reactive site peptide bond, which connects residues
designated as P and P
`(8) . This bond
serves as substrate for the cognate proteinase. In all free inhibitors
whose structure has been determined, the P
residue is
highly exposed to solvent(7, 9) . In complexes, the
reactive site peptide bond remains intact and planar, but the carbonyl
carbon atom of the P
residue is in exceptionally close
contact with the OG atom of the catalytic Ser residue of the
enzyme(9) . Most importantly, the P
side chain
becomes imbedded in the S
pocket (7, 9) of
the enzyme. This pocket is often called the primary specificity pocket
of serine proteinases. While the interaction of P
with the
enzyme is energetically the most important, about a dozen of the
inhibitors' residues interact both by main chain-main chain
hydrogen bonds and by specific side chain-side chain interactions.
Collectively, the interactions of all the contact residues, other than
P
, are energetically dominant. They ensure that the same
peptide bond in the inhibitory domain acts as the reactive site for
numerous different cognate enzymes(10) . More importantly, they
ensure that, in inhibitor mutants, substitutions of the P
residue do not shift the reactive site peptide bond and that the
mutant P
side chain is still imbedded in the S
cavity of the enzyme, even if that interaction alone is
energetically adverse rather than favorable(
)(11) .
Turkey ovomucoid third domain, OMTKY3, ()(Fig. 1)
is a widely studied ``standard mechanism,'' canonical
inhibitor of serine proteinases. With its P
Leu residue, it
is a powerful inhibitor of many chymotrypsins, elastases, subtilisins,
and two of the many Pronase components, Streptomyces griseus proteinase A (SGPA) and proteinase B (SGPB)(10) . All of
these enzymes have predominantly hydrophobic S
pockets and
prefer substrates and inhibitors with hydrophobic P
residues. We have recently acquired 25 different P
variants (all 20 coded and 5 noncoded) of X
OMTKY3 and measured their association constants, K
, at pH 8.3 with six enzymes, all of
which are members of the set listed above(18, 19) . In
a parallel study, Huang and James
undertook to obtain high
resolution three-dimensional structures of all 20 coded X
variants of OMTKY3 complexed with SGPB. Many of
these structures, which crystallized near pH 6.5, especially Glu
OMTKY3, are already in hand.
The binding of
Glu
to the S
specificity pocket of SGPB is
similar to the binding of Leu
(11, 14, 15) . The interpretation of the K
(at pH 8.3) and the x-ray data (at pH
6.5) requires knowing whether the Glu
residue is
protonated (Glu
) or deprotonated (Glu
) in
complex at these values. When appraised of our results that the pH 6.5
structure was of the protonated form, Glu
, Huang and
James
raised the pH of their crystals to 10.7. The global
structure of the complex was unaltered, but clear evidence was obtained
for Glu
rather than Glu
.
Figure 1:
Amino acid sequence of
OMTKY3(12) . For historical reasons the numbering starts from
residue 6 rather than 1. The 12 consensus contact
residues(13) , which are in contact with SGPB (14, 15) , chymotrypsin(16) , and human
leukocyte elastase(17) , are highlighted in black. The reactive site peptide bond between the P residue Leu
and P
` residue is indicated
by an arrow. It is the Leu
residue that was
replaced in the Glu
, His
, and
Gln
variants.
The pH dependence of log K for three
ovomucoid third domain variants, namely ionizable Glu
OMTKY3 and His
OMTKY3 and a nonionizable Gln
OMTKY3, as a reference, is shown in Fig. 2. The behavior
of log K
for the last case is extremely simple. It
can be rationalized by the pK of His
in the
catalytic triad of SGPB being 6.8 in the free enzyme and shifting to a
very low value in the enzyme inhibitor
complex(26, 27) . It is worth noting that while the
assignment of the pH dependence of Gln
OMTKY3-SGPB
association is likely correct and interesting, it is not essential to
this paper. All that is required is that the division of K
(Glu
) and (His
) by K
(Gln
) eliminates the effect of all
residues other than the P
residue. For Glu
and
His
variants, the pH dependence is more complex, involving
a combination of the effects seen in the Gln
variant and
the pK perturbation of the P
residue by complex
formation. To separate the effects, consider,
Figure 2:
pH dependence of log Kof SGPB with Gln
OMTKY3
(
), Glu
OMTKY3 (
), and His
OMTKY3
(
). The values were determined by extensive modification of the
Green and Work method (22) as
described(21, 23, 24, 25) .
where K is at arbitrary pH,
K
is at very low pH where all the groups in
the complex, inhibitor, and enzyme are fully protonated. Q
, Q
, and Q
are the protonation state partition functions, often called
binding polynomials(28) , but, in this case, probably better
referred to as proton release
polynomials(29, 30, 31) . is a
general equation for the pH dependence. However, in the free inhibitor,
the P
residue is fully exposed and does not interact with
other residues(6, 7, 9) . Therefore, for
Glu
OMTKY3 (or His
OMTKY3) variants we can
write,
where K is the ionization constant of
P
in the free inhibitor and Q
* is the
proton release polynomial for all the other ionizable residues of the
inhibitor. In this approximation, Q
* is obtained
from a nonionizable P
variant such as Gln
OMTKY3. Similarly, since the P
side chain in the complex
with SGPB is insulated in a hydrophobic S
cavity
(11) , it seems plausible to write,
where K is the ionization constant of
the P
residue in the complex. Simple division now yields as
follows.
The derivation of this relation is based on a simple assumption
that the ionizable P residue is not interacting with any
other ionizable group in the free inhibitor or in complex. This
assumption is supported by the published x-ray structure of ovomucoid
third domains (7) and by the unpublished structure of
Glu
OMTKY3 in complex with SGPB.
For plotting
and fitting of the data the logarithmic form of this equation has been
used,
where R is the equilibrium constant ratio
of the fully protonated to the nonionizable residue and
pK
and pK
are the pK values in the free inhibitor and in the complex with SGPB. The
plots of log R versus pH are shown in Fig. 3. The
excellent leveling off for the His
and Glu
curves, both on the low and high pH side, clearly indicates that,
around pH 3, the P
side chain in complex and in the free
inhibitor is predominantly in the His
and Glu
forms and, near pH 10, it is predominantly in His
and
Glu
forms, both in the free inhibitor and in complex.
The plots in Fig. 3are fitted by nonlinear least squaring (32) using pK
, pK
,
and R
as fit parameters. For Glu
pK
is 4.46 ± 0.05, pK
is 8.74 ± 0.06, and R
is 13.9
± 1.3. The large 4.3-unit pK shift shows that (a) both in the crystal structures and in measurements at pH
8.3, the side chain of Glu
is predominantly in the
Glu
form, (b) binding of Glu
is 16,000
times stronger than binding of Glu
, and (c)
binding of Glu
is 14 times better than binding of Gln. A
simple illustration of the pK shift of Glu in Glu
variant upon complex formation with SGPB and the associated
changes in K
are shown diagrammatically in Fig. 4. For His
, pK
is 6.63
± 0.08, pK
is 4.31 ± 0.07, and R
is 0.027 ± 0.003. The downward shift of
the His
pK is consistent with avoidance of a
charged form in the S
cavity, albeit now only by a factor
of 160. The binding of His
, in its neutral form, is 4.5
times better than that of Gln
; this was already known from
the pH 8.3 measurements(18, 19) . Note the striking
and unforced agreement between pK
of both side
chains with the expected or model values. The excellent fit of the
curves to and the clear evidence that measurable
association still takes place at the low pH limit of Fig. 3for
the His
variant, and at the high pH limit for the
Glu
variant, argue for the validity of the model. The
correctness of this treatment is also strongly validated by the ratio
of two nonionizable side chains, i.e. (K
(Leu
))/(K
(Gln
))
being independent of pH in the pH range 4-8.3 (Fig. 3).
Figure 3:
pH dependence of log R, where R is the ratio of (K(Glu
))/(K
(Gln
))
(
), (K
(His
))/(K
(Gln
))
(
), or (K
(Leu
))/(K
(Gln
))
(
). The curves in the case of Glu
and
His
variants connecting the points are the best fits to .
Figure 4:
Schematic representation of pK shift of Glu in Glu
OMTKY3 upon complex
formation with SGPB. While the value of K
at pH 10 for (Glu
)
is an
experimentally measured number, the value of K
at pH 10 for (Glu
)
was calculated
by multiplying the K
value of the
Gln
variant at pH 10 by the ratio of K
(Glu
)/K
(Gln
)
in fully protonated form, i.e. by R
.
The values of K for variants with ionizable
side chains that were reported (18, 19) at pH 8.3
refer to the equilibrium mixtures of the ionized and unionized forms of
the P
side chain both in the free inhibitor and in the
complex. In the specific case of Glu
variant interacting
with SGPB, the free side chain is overwhelmingly Glu
,
while the bound side chain is largely Glu
. It is of
interest to calculate the K
values for Glu
in the free inhibitor binding to form a complex with Glu
and for Glu
in the free inhibitor forming a
complex with Glu
. The constants are K
(Glu
)
1
10
M
and K
(Glu
)
6
10
M
(see Fig. 4). The K
(Glu
) is the
fourth strongest of K
values (after Leu
(5.6
10
M
),
Met
(2.7
10
M
), and (Asp
)
(2.0
10
)) of all 20 coded amino acid residues. At low
pH (pH 4.5 or lower), the Glu
variant is one of the most
effective inhibitors of SGPB. It is possible that the frequently seen
P
Glu residues in natural protein inhibitors of serine
proteinases serve not only to inhibit glutamic and aspartic
acid-specific enzymes such as Glu-specific S. griseus proteinase (33) but also at low pH values to inhibit the
widely distributed enzymes with hydrophobic S
pockets.
While the method is fairly labor intensive, we believe that it could
be readily extended to more, possibly all, ionizable side chains (Asp
and Lys seem particularly good) and to more enzymes with hydrophobic
S pockets, e.g. SGPA and chymotrypsin. Extension
to enzymes, such as trypsin or Glu-specific S. griseus proteinase, with ionizable residues in their S
pockets
may require more complex interpretation.
Ionizable side chains in
proteins in their protonated form are either uncharged (Asp, Glu, Cys,
Tyr) or cationic (His, Lys, Arg)(34) . Ionization of uncharged
acids yields charged products. Therefore the large upward pK shift, seen upon addition of organic cosolvents to such acids, is
expected(35, 36, 37) . On the other hand,
ionization of cationic acids involves a transfer of charge from the
cationic reactant to the hydronium ion, which is one of the products.
Therefore, cosolvent effects on pK are anticipated to be, and
are, small and erratic(35, 36, 37) . Burial
in a protein differs from transfer to an organic solvent, as the
hydronium ion resides in an aqueous solution; only the side chain is
buried. As a result, opposite effects, about half as great in absolute
value as those observed in the transfer of an uncharged acid to an
``equivalent'' organic solvent, should be expected if the
S pockets were purely isotropic. Clearly, they are not, and
dipole and charge effects may further affect the observed values.
Since this communication was first submitted, we have determined the
pH dependence of the interaction between SGPB and Asp OMTKY3 and found that pK
is 4.40 ±
0.10 and pK
is 9.26 ± 0.10. Therefore, the
shift is 4.9 units, somewhat greater than for Glu
.
Recently (38) it was shown that Asp
in thioredoxin
has a pK of 7.5 in the oxidized form and greater than 9 in the
reduced form, another huge pK shift for an Asp residue. Huang
and James
completed their high resolution structures of
Asn
OMTKY3 at pH 6.5 and of Asp
,
Glu
, and Gln
variants at pH 6.5 and 10.7. An
extended joint manuscript is now readied for publication. (
)