Download Compressibility gives new insight into protein dynamics and enzyme

Biochimica et Biophysica Acta 1595 (2002) 382^386
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Review
Compressibility gives new insight into protein dynamics
and enzyme function
Kunihiko Gekko *
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama,
Higashi-Hiroshima 739-8526, Japan
Received 16 November 2001; accepted 19 November 2001
Abstract
The adiabatic compressibility of enzyme is largely influenced by binding of coenzyme and substrate, due to the changes in
atomic packing. Amino acid substitution also induces large changes in compressibility parallel to enzyme activity. These
results demonstrate that a small alteration of local structure by ligand binding and mutation is dramatically magnified in the
flexibility of protein molecule to affect the function. Compressibility gives new insight into protein dynamics and enzyme
function from the aspect of atomic packing or cavity which cannot be obtained by other techniques. ß 2002 Elsevier
Science B.V. All rights reserved.
Keywords: Adiabatic compressibility; Amino acid substitution; Cavity; Enzyme function; Ligand binding; Protein dynamics
1. Introduction
Compressibility is not only a basic physical quantity to analyze the pressure e¡ects on protein structure, but also it gives new insight into protein dynamics which contributes to biological function of
protein molecules. According to the X-ray crystal
structure, globular proteins have a precisely de¢ned
compact structure and all atoms in the molecule
seem to be closely packed as well as the crystalline
amino acids. However, there are many packing defects or cavities in the interior of a protein molecule,
which cause internal motions or £exibility response
to thermal or mechanical forces. Such cavities are
easily compressed by pressure, so compressibility is
* Fax: +81-824-247387.
E-mail address: [email protected] (K. Gekko).
an important measure of protein £exibility or volume
£uctuation. According to statistical thermodynamics,
the volume £uctuation NVrms of a protein with volume Vp is related to its isothermal compressibility LT
[1]:
N V rms ¼ ðkTV p L T Þ1=2
ð1Þ
where k is the Boltzmann constant and T the absolute temperature.
Measurement of compressibility is possible, in
principle, only for proteins in solution. Although volume £uctuation is directly linked to isothermal compressibility, most compressibility studies of proteins
have been of adiabatic compressibility because of the
accuracy and technical convenience [2^5]. The partial
speci¢c adiabatic compressibility Ls ‡ is de¢ned as the
change in the partial speci¢c volume v‡ with pressure.
Since the constitutive atomic volume of a protein
may be regarded as incompressible, the experimen-
0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 3 5 8 - 2
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K. Gekko / Biochimica et Biophysica Acta 1595 (2002) 382^386
tally determined Ls ‡ can be mainly attributed to its
cavity vcav and to changes in hydration vvsol [3],
L s ¼ 3ð1=v ÞðD v =D PÞ
¼ 3ð1=v Þ½D vcav =D P þ D v vsol =D PŠ
ð2Þ
The cavity contributes positively and hydration negatively to v‡ and Ls ‡, then Ls ‡ can be positive or
negative depending on the magnitude of both terms.
Most globular proteins have a positive Ls ‡ value of
0^14 Mbar31 at 25‡C, indicating the large contribution of cavity overcoming the hydration e¡ect [3].
The volume £uctuation is in the range from 30 to
200 cm3 mol31 , which correspond to only 0.3% of
whole protein volume. But if concentrated in one
area, it would produce su⁄cient cavities or channels
in the protein molecule to allow penetration of solvent or probe molecules. Recent studies on the e¡ects
of ligand binding and amino acid substitution on Ls ‡
demonstrate that a small alteration of local structure
is dramatically magni¢ed in the overall protein dynamics to a¡ect the enzyme function [6^9].
2. E¡ects of ligand binding
The enzyme^ligand complex formation generally
proceeds in three states: formation of net bonds between enzyme and ligand, changes in hydration of
the interacting species, and conformational changes
of the enzyme. The solute-solute interaction in water
should accompany dehydration of each solute molecule, resulting in the increase in v‡ and Ls ‡. However,
the observed changes in both parameters by ligand
binding are not necessarily positive. A typical example is lysozyme^inhibitor complex formation. The v‡
and Ls ‡ values of lysozyme decrease by addition of
N-acetyl-D-glucosamine oligomers, in the order of
monomer s dimer s trimer [6]. Assuming that the decrease in Ls ‡ is ascribed to the protein moiety only,
the volume £uctuation is estimated to decrease by
about 3%, 7%, and 14% on binding the monomer,
dimer and trimer, respectively. The X-ray crystallographic study shows that the inhibitors are bound in
the cleft of the active site of lysozyme. The e¡ects of
this ‘induced-¢t’ type interaction would propagate
throughout the entire protein molecule to enhance
the internal atomic packing, resulting in the de-
383
pressed £uctuation of the protein molecule. The hydrogen-exchange rate of lysozyme is also known to
decrease in the same order of these oligomers [10]. It
is noteworthy that the volume £uctuation changes
comparably to the kinetically determined £uctuation.
More pronounced changes in Ls ‡ and v‡ are found
on ligand binding of Escherichia coli dihydrofolate
reductase (DHFR). DHFR catalyzes the reduction
of dihydrofolate (DHF) to tetrahydrofolate (THF)
with the aid of coenzyme NADPH in the kinetic
reactions cycling through ¢ve intermediates,
DHFRWNADPH, DHFRWNADPHWDHF, DHFRW
NADPþ WTHF, DHFRWTHF, and DHFRW NADPHW
THF [11]. This enzymatic function is strongly repressed with an inhibitor, methotrexate (MTX).
The X-ray structure of a DHFRWNADPHWMTX ternary complex is shown in Fig. 1 [12]. The Ls ‡ of these
kinetic intermediates is shown as a function of the
reaction coordinate in Fig. 2 [7], where the transient
state DHFRW NADPHWDHF is substituted by a complex DHFRWNADPHWMTX since the X-ray structures of both complexes are quite similar. Evidently,
the £exibility of DHFR changes alternatively by
binding or releasing the coenzyme and substrate:
the transient state DHFRWNADPHWDHF is expected
to be most £exible and the state DHFRWNADPþ W
THF most rigid in the intermediates. As illustrated
in Fig. 3, the number, size and distribution of cavities
also change on ligand binding, parallel to the
changes in partial speci¢c volume (vv‡) and compressibility (vKs = vv‡Ls ‡). On binding of these ligands,
the total cavity volume is largely in£uenced ( þ 40%)
but the solvent accessible surface area only slightly
decreases (at most 6%), indicating that the cavity
plays dominant role in ligand-induced compressibility change overcoming hydration e¡ects. Recent high
pressure NMR study of folate-bound DHFR reveals
that major conformation changes by pressure occur
in the NADPH binding site and the £exible loops
[13].
3. E¡ects of amino acid substitution
How does the single amino acid substitution a¡ect
the £exibility of overall protein structure? A key to
answer this problem is found in the mutation e¡ects
on Ls ‡ of two E. coli enzymes, aspartate aminotrans-
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K. Gekko / Biochimica et Biophysica Acta 1595 (2002) 382^386
Fig. 2. The Ls ‡ level of the kinetic intermediates of DHFR as a
function of the reaction pathway [7].
Fig. 1. The structure of a DHFR^MTX^NADPH ternary complex taken from Sawaya and Kraut [12]. Positions Gly67,
Gly121, Ala145 (mutation sites) and Asp27 (catalytic site) are
indicated by arrows.
ferase (AspAT) and DHFR. AspAT has two identical 45 kDa subunits and the catalytic site is formed
between the large and small domains in each subunit.
Six mutant enzymes at Val39, which is located at the
gate of the substrate binding site, have evidently different Ls ‡ values [8]. The observed positive linear
correlation between Ls ‡ and van der Waals volume
of the introduced amino acids suggests that the bulkiness of the side chain at position 39 makes the protein structure more £exible due to the overcrowding
e¡ect in atomic packing. The catalytic e⁄ciency of
AspAt is also modi¢ed by these mutations, where
major changes occur on Km but not on kcat . Interestingly, there is a linear correlation between log (kcat /
Km ) and Ls ‡, showing that the amino acid substitution at Val39, which increases £exibility of overall
structure, makes the enzyme more active.
The enzyme activity of DHFR is known to be
in£uenced by mutations at Gly67, Gly121, and
Ala145, which are located at the center of three different £exible loops (see Fig. 1), where Km is only
slightly in£uenced but kcat remarkably changes [14^
16]. This is very interesting because it is unlikely that
the mutation sites directly participate in the enzyme
reaction: K-carbons of Gly67, Gly121 and Ala145
î
are separated respectively by 29.3, 19.0 and 14.4 A
from the catalytic site Asp27, and at shortest by 8.5,
î from the NADPH molecule. Fur10.6 and 29.2 A
thermore, double mutants at Gly67 and Gly121,
î,
whose K-carbons are mutually separated by 27.7 A
show non-additive e¡ects on the function [17]. These
¢ndings suggest that these mutations would alter the
Fig. 3. The changes of number (n), position and total volume (vVcav ) of cavities in some kinetic intermediates of DHFR [7].
vKs = vv‡Ls ‡: cm3 g31 Mbar31 .
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K. Gekko / Biochimica et Biophysica Acta 1595 (2002) 382^386
385
local structure due to ligand binding and amino acid
substitution is dramatically magni¢ed in the overall
protein dynamics, possibly via ‘stepwise atomic reconstruction or long-range cooperative e¡ect’, leading to modi¢cation of catalytic activity of enzymes.
Compressibility study in combination with X-ray
crystallography and high-pressure NMR would be
fruitful for elucidating the structure^£exibility^function relationship of enzymes.
References
Fig. 4. Plots of Km and log (kcat /Km ) against Ls ‡ of DHFR mutants [9].
£exibility of the entire protein molecule. In fact,
these mutations induce large changes in v‡ (0.710^
0.733 cm3 g31 ) and Ls ‡ (31.8^5.5 Mbar31 ) from
the corresponding values of wild-type enzyme
(v‡ = 0.723 cm3 g31 , Ls ‡ = 1.7 Mbar31 ) [9]. As shown
in Fig. 4, there is a de¢nite correlation between Ls ‡
and Km or log (kcat /Km ), indicating that the structural
£exibility positively contributes to the enzyme function, as is the case of AspAT, through an enhanced
catalytic reaction rate and in part due to increased
a⁄nity for the substrate. It is important that the
£exibility-mediated modi¢cation of enzyme activity
can be derived by the mutation at the loop regions
far from the catalytic site because this gives a new
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(K. Katayanagi et al., to be published). These results
demonstrate that the e¡ects of mutation extend to a
long range and the mutation-induced Ls ‡ change is
dominantly ascribed to modi¢cation of internal cavities.
As shown here, compressibility gives new insight
into protein dynamics and enzyme function from the
aspect of atomic packing or cavity which cannot be
obtained by other techniques. A small alteration of
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