TIBS 17 - MARCH 1992
REVIEWS A RIGOROUS ~ S T of our understanding of structure-activity relationships in proteins is the use of those relationships in altering the function of a protein in a prescribed fashion. The field of protein engineering has progressed to the point where the rational design of proteins is now providing an avenue for the development of proteins as reagents with a practical use. This is especially true of proteins whose activities and/or structures are dependent upon coordinating metal ions. Our knowledge of such metalloproteins has expanded, allowing basic rules governing protein-metal interactions to be formulated and applied in engineering metalloproteins for specific purposes. A variety of metal ions are known to interact with a large number of proteins required for a host of physiological processes L2. The role of the bound metal ion in these metalloproteins is varied but, in all cases, metal ions possess features that make them useful as cofactors in proteins 3. Metal ions associated with proteins have been implicated in substrate binding, catalysis, protein folding and stabilization. Metal ions play important roles in mediating protein-protein and protein-DNA intermolecular interactions4,s and have also been implicated as physiological regulators of enzymatic activity6,7. Fundamental principles based on protein-metal interactions of naturally occurring metalloproteins can now be put to use in creating metalloproteins with specific characteristics. Of particular interest is the design of metal iondependent regulatory sites in enzymes, since the ability to control the activity of an enzyme by manipulating the availability of metal ions in the external media may have useful clinical or industrial applications.
Chemical principlesof metal bindingsites A survey of metalloproteins of known three-dimensional structure has identJ. N. Higaki and C. S. Craik are at the " Department of PharmaceuticalChemistryand R. J. Flettorick and C. S. Craik are at the Department of Biochemistryand Biophysics, Universityof California, San Francisco, CA 94143, USA.
100
Engineered metalloregulation in enzymes
Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.
ified important features of metal-protein with metal ions, the infrequent occurinteractions that can be used in the rence of a free sulfhydryl group in prodesign of a metal-dependent regulatory teins makes His the primary amino acid siteL Since it is the unique electronic residue involved in metal binding ~°," properties (charge, coordination number and the one most commonly used in and geometry) of certain metal ions engineering metal binding sites. that make them useful in biological sysA method for the prediction of metal tems 2,8,one of the first considerations is binding sites in proteins has been prothe type of metal ion to be used. Among posed 12 that describes important structhe most common metal ions in naturally tural features of a protein environment occurring metalloproteins are first row that favor metal binding. However, the transition metal ions (Zn 2+, Cul+/2+, orientation of the amino acid residues Fe2÷/3+) with typical coordination num- serving as ligands must also compbers ranging from 4 to 6. These ions lement the preferred coordination gehave favorable coordination geometries ometry of the bound metal ion 2. In a that enable them to form stable interac- number of metalloproteins, the presence tions with the amino acid side chains of of His side chains that are able to adopt proteins. Although the stability of preferred orientations provides clues as metal-ligand interactions is determined to how adjacent imidazole side chains in a large part by features of the metal can form stable metal binding sites ~3. ion itself, it is also dependent on both When two or more His side chains the availability and the conformation of are available [o coordinate a metal ion, associated protein ligands. certain unique spatial orientations of Amino acid residues that function as these side chains can favor enthalpic good metal ligands are typically those and entropic effects leading to tighter that contain electron-donating atoms binding (chelation) of a metal ion9. For (S, O or N) 9. Although this group example, a His-X-X-X-His motif in an includes those containing unionizahle m-helix of a number of naturally-occurgroups (Trp, Ser, Met) the strongest ring proteins possesses two correctly interactions typically involve amino positioned imidazoles that together can acid side chains with ionizable groups chelate a metal ion. Such a structure (Asp, Glu, Cys, His). Since it is the can be engineered into proteins to engreater basicity of the functional group hance their meta! binding properties ~4. (higher pKa) that makes it the better By using these and other principles of ligand, the order of copper binding metal binding derived from both theorability for these amino acids is etical work and empirical observations Cys > His > Asp/GIu. Basic amino acid on metalloproteins of known strucresidues Lys and Arg rarely participate tures, strong metal binding sites are in metal binding and despite the fact now being engineered into proteins for that Cys forms such strong interactions a variety of purposes. © 1992,ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00
TIBS 17 - MARCH 1992
Within the past few years, interest in the design of metal binding sites in proteins has been expanding rapidly as more applications of engineered metal binding sites develop, many of which have recently been reviewed ~s. For example, metal binding sites have been introduced into proteins to stabilize them against denaturation or proteolytic degradation. They have also been engineered into proteins to assist in their purification ~5-~8, crystallization ~9, and X-ray crystal structure determination 2°. lverson et al. 2~ have reported the design of a metalloantibody that contains a His coordination site for metals in the antigen binding pocket. This is an important step forward in the design of antibodies that could catalyse redox and hydrolytic reactions. An exciting development in catalytic antibodies was recently described that involves the isolation and initial characterization of an antibody capable of catalysing porphyrin metallation 22. Further analysis of enzymes generated in this fashion should contribute unique and insightful information to the database of metalprotein interactions.
lated more precisely. In addition to enhancing the zd + control one has over the enzymatic reaction, this 2.1 2.1 mechanism of enzyme ~.~ .~ 102° %.. /"'~NE' 'Ne7 \ regulation also permits a unique analysis of structure-activity relationships in the engineered protein. Several proteins have been engineered so that their activity can be regulated in vitro by exploiting disulfide bonds 25,26,salt bridges 27 and electrostatics 28,~9. Based on the database of information regarding Figure 1 metalloprotein structure An ideal metal binding site. The side chains of His142 and His146, along with Glu166 (not shown) comprise and on the natural role of the zinc binding site of thermolysin (file 2TLN, Brookmetal ions in regulating enhaven Protein Data Bank). This naturally-occurring zyme activity, metal bindmetal binding site illustrates the orientation of the His ing sites are presently side chains required for strong metal binding. The imidabeing engineered into enzole side chains of His142 and His146 are positioned zymes for enhanced regu102" relative to each other and are situated 2.1 A from the bound zinc ion. The corresponding Ca.of these two lation. One of the first examino acid residues are less than 13 A apart, This amples of an engineered metal binding geometry was used as a template for regulatory site involving locating potential metal binding sites in proteins of the binding of a metal ion known three-dimensional structure. was that reported for staphylococcal nuclease 3°. Metal.dependent enzyme regulation The ability of sulfhydryl groups to co- near the enzyme active site could result A novel application of an engineered ordinate metal ions was exploited in in a local conformational change upon metal binding site in proteins is the redesigning staphylococcal nuclease so binding the metal ion that would alter introduction of such a site into a pro- that a free cysteine was exposed at the the catalytic properties of the enzyme. tease for regulation of enzyme activityz3. base of the substrate binding pocket. Co- By making the metal binding site depenThis mechanism of metalloregulation ordination of this sulfhydryl group with dent upon an active site amino acid, relies on the binding of metal ions to either organic mercurial compounds or enzyme activity provides a rapid evaluinduce local structural changes in an copper occluded the substrate binding ation of the original design principles. enzyme that result in altered catalytic pocket rendering the enzyme inactive. The three-dimensional structure of properties. Creation of such a metal By removing the bound metal ion with an enzyme can be searched for suitable switch is based on the three-dimensional strong chelating agents, the pocket was sites that, when substituted with His structure of the enzyme, the chemical cleared and the enzyme activity was amino acid residues, would form a mechanism and many of the concepts restored 3°. Although powerful, this strong metal binding site capable of generally accepted regarding metal ion method may not always be possible in altering the catalytic activity of an encoordination in proteins. The informa- proteins that contain naturally occur- zyme. The unique ability of an ention gained from these experiments may ring cysteines or disulfide bonds since gineered metal binding site to function lead to a general method for engineering the presence of a free sulfhydryl might in regulation relies on the ability of the a metal regulatory site into any enzyme. interfere with protein folding in certain bound metal ion to alter the conforThe regulation of enzyme activity is expression systems. Futhermore, the mation of amino acid residues critical an~essential feature of all physiological oxidation state of cysteine may be dif- for activity. If sites for His substitutions processes. In vitro, the activity of an en- ficult to control in the presence of are chosen that would provide a potenzyme can be regulated by manipulating metal ions, making histidines an attrac- tial metal binding site involving the environmental factors such as pH, tem- tive alternative to cysteines for en- active site His, the coordination of this perature, ionic strength, substrate con- gineered metal chelation. critical His to a bound metal ion would centration, and exposure to specific reversibly inactivate the enzyme by inhibitors. Manipulating the in vitro Histidines as targets for engineered preventing its participation in catalysis. activity of an enzyme has led to a metal.dependent regulation The potential amino acid pairs of the wealth of knowledge concerning the The approach taken in introducing a metal switch need to satisfy five importrole of enzymes in biochemical reac- metal-dependent regulatory site into an ant criteria based on our current undertions and the mechanisms employed to enzyme is based on the ability of imida- standing of metal binding sites: carry out their specific functions 24. zoles 3~ and of properly oriented His With the advent of recombinant DNA amino acid side chains 9,32 to chelate (1) The side chains should be solvent technology came the ability to engineer transition metal ions. Strategic placeaccessible for exogenous metal the enzyme itself so that it can be regu- ment of a histidine amino acid residue binding.
/
101
TIBS 17 - MARCH 1992
(2) The distances between the mcarbons of the unsubstituted side chains should be less than 13.0 ]~ to allow the corresponding imidazole side chains to chelate the metal. (3) The His side chains should be any one of five favorable rotational conformers 33 and still coordinate the metal ion. If the torsional angles of the side chains correspond to an unfavorable free energy, then some free energy gain that derives from chelating the metal will be lost. However, the metal sites in natural proteins are often coordinated to His side chains which are not favorable rotamers suggesting that this is not a stringent requirement. (4) All atoms of the substituted His side chains should be further than 3.0 A from other atoms of the pro-
(5)
rein unless a hydrogen bond could be formed. This design criteria would prevent any steric overlap that might cause distortions in structure. Binding of the metal ion should induce a conformational change that could alter the catalytic properties of the enzyme.
Computer programs that employ these or similar criteria are available or are currently being developed (Biosym Technology). A quintessential example of a metal binding site is found in thermolysin (file 2TLN, Brookhaven Protein Data Bank; Fig. 1). Two histidines help coordinate the Zn 2÷ in this metalloprotease. The cis orientation of the histidines liganded to the Zn 2÷ places them at an angle of approximately 102°. The
Figure 2 Schematic representation of the metal switch in trypsinR96H. The Ca positions of the active site region are shown along with the modeled coordination complex formed between a single Cu2+ ion, His96 and His57 of trypsinR96H. The coordinates for the side chain of Arg96 were replaced with the coordinates for a His and the torsional angles of His57 and His96 were adjusted manually to superimpose with His142 and His146 of thermolysin. His142 and His146, along with Glu172, bind the Zn 2+ ion in the active site of thermolysin and represent a functional coordination complex. After adjustment of the X2 angle for the His57 side chain by 50 °, a root mean square superposition of less than 0.4/~ was achieved for the His atomic coordinates of thermolysin and trypsinR96H. This coordination complex places the Cu2÷ ion at a position that is simultaneously 2.2 A (subtending an angle of 95") from the N¢ nitrogen atom of both His side chains. Subroutines from the program Insight (Biosym Technologies, San Diego) were used to perform the alignments and torsional angle adjustments of the side chains. His57 is normally positioned in the active site of trypsin where it is within hydrogen bonding distance to the side chains of Aspl02 and Ser195. These three residues cor~stitute the functional catalytic triad of trypsin. The N¢ atom of the His57 side chain is expected to move 6 A (curved arrow) on coordination with a Cu2÷ ion that binds to His96. The arrows pointing from the N~ atoms of the two imidazole groups to the Cu2÷ ion represent coordinate covalent bonds. Removal of the bound Cu2÷ ion allows the His57 side chain to rotate back into the active site thereby reestablishing the functional catalytic triad. A space-filling version of this figure is shown on the front cover of this issue.
102
distance between the metal and the coordinated nitrogen of the imidazole is approximately 2 A. This configuration exemplifies the His-metal-His template that was used to search the threedimensional structure of a protein for potential metal binding sites.
Metal-dependent regulation of trypsin Although the criteria described above could be met in various proteins, trypsin was a particularly attractive enzyme to test the design principles. Trypsin catalyses the hydrolysis of ester and amide bonds carboxy-terminal to the c(-carbon of Arg and Lys amino acid residues. It is a member of a large and well-studied family of serine (Set) proteases that all have a nucleophilic Set amino acid residue at the active site. In the catalytic mechanism of Ser proteases, His57 serves initially as a general base to increase the nucleophilicity of the essential Ser and subsequently as a general acid to donate a proton to the leaving group 24,34. Because it is critical for catalytic activity, repositioning of the His57 side chain away from the active site will disrupt the conformation of the catalytic triad, rendering the enzyme inactive. To reposition the imidazole of His57, a second His was placed in the vicinity of His57, thereby forming a two-histidine metal binding site 23. Based on the five criteria listed above and using the coordinates for rat trypsin (file 2RTM, Brookhaven Protein Data Bank), nine pairs of potential His pairs satisfy the first four criteria set by the search algorithm; however, only His57 together with a His substituted for Arg96 satisfy the fifth criteria. Computer modeling studies indicated that, in the presence of Cu2+, the side chain of His57 can rotate 90 ° ab6ut the C~-Cp bond forming a His96-His57-Cu 2+ coordination complex that can be superimposed onto the natural metal-chelating site of thermolysin. The resultant metal switch is shown in Fig. 2; His57 is shown both in catalytic register within the catalytic triad, and out of catalytic register when coordinated to a single Cu2÷ion. Having identified a potential site (position 96) for His substitution, sitedirected mutagenesis of rat anionic trypsin was used to substitute Arg96 with a His (trypsinR96H) 23. The velocity of the His96-modified enzyme could be altered by the presence of metal ions as dramatically demonstrated in Fig. 3. The kinetic parameters of trypsinR96H in comparison to trypsin (Table I) also
TIBS 1 7 - MARCH 1992
show that the enzyme closely resembles trypsin in the absence of Cu2÷, but has a compromised kca t value in the presence of 0.4 mM Cu2÷. Removal of the bound Cu2+ restores the variant to its active state. This is a textbook case of non-competitive inhibition with a K~ of 21 ~M for copper, 49 tim for nickel and 128 laM for zinc23. The order of binding is, as expected, based on the binding of those metal ions to imidazole. The Lewis acidity of the metal governs the order of binding to the ligand; however, the absolute value of the association constant is critically dependent upon the geometrical constraints of the metal binding site imposed by the protein scaffold. The three-dimensional structure of the engineered metal switch in the presence and absence of metal should provide a structural framework for analysing its affinity for transition metals. The structure will also be invaluable for creating subsequent metal binding sites with higher affinity and with greater selectivity. Serine/metallo-protease link?
(a)
J
B
C D
0 I
E
-~
,
I
~
I
I
~
I
(b) 60
-R,
40
20
- an evolutionary
Wild-type trypsin was shown to be partially and reversibly inhibited by binding of a single metal ion at very high concentrations of Cu2÷ (Ref. 23). This was consistent with the observation that the Asp102-His57 couple of trypsin is inhibited by binding of a silver ion35. Other serine proteases have also been reported to be inhibited by metal ions. A few notable examples are the inhibition of catalytic activity of et-chymotrypsin and (~-lytic protease3E The autocatalytic activation of the zymogen form of the ~-subunit of nerve growth factor has also been shown to be controlled by zinc 37. Although the mode of metal interaction has not been characterized for these serine proteases, it is possible that they all have a similar mechanism of metal binding. Thus, Asp~His couples that are capable of binding metal ions might naturally play a role in protein folding or in enzyme regulation. The binding of a metal ion at the Asp102-His57 couple in the catalytic triad of Ser proteases is structurally similar to the way in which metal ions are coordinated as a component of the catalytic triad of zinc metalloproteases such as carboxypeptidase A38 and thermolysin39. The metal binding Asp-His couple of serine proteases may be an evolutionary structural motif linking the active sites of serine proteases to zinc
[]
A
0"-
t
0
,
50
I
~
i 200
I
100
150
[ Z - G P R - A M C ] (pM) Figure 3 Velocity versus substrate concentrations for trypsinR96H (a) and trypsin (b) at various concentrations of Cu2÷. The activity of 1.25 nM enzyme was monitored fluorometrically at 25°C using varying concentrations of the tripeptide substrate, Z-GPR-AMC. Assay conditions were as follows: 1 mM Tris, pH 8.0 containing 1 mM CaCl2, 10 mM NaCI and (A) 0 mM CuCl2; (B) 0.02 mM CuCl2; (C) 0.04 mM CuCl2; (D) 0.1 mM CuCl2; (E) 0.2 mM CuCl2. The excitation wavelength was set at 380 nm and the emission wavelength was set at 460 nm. (a) The maximum velocity for trypsinR96H at high Z-GPR-AMC concentrations (200 IJM) reached an increasingly lower limit at higher concentrations of Cu2÷. (b) The activity of 1.25 nM wildtype trypsin is relatively unaffected by Cu2÷ ions using the same conditions described in (a).
metalloproteases 4°. In the serine proteases, the Asp-His couple might have recruited an activated serine, resulting in the catalytic triad. The metallopro-
teases, alternatively, could have used the Asp-His couple to incorporate a metal ion at the active site of the enzyme.
Table I. Kinetic parameters a of trypsin and trypsinR96H
Enzyme
Trypsin TrypsinR96H
CuCl2 (mM)
kcat (min-1)
Km (I.IM)
kcat / K m (minlpM-1)
(pM)
0.0 0.4
3345 2510
14.1 16.7
237.3 150.4
1 600
0.0 0.4
2464 495
7.5 26.0
331 19.0
21
K i
aproteolytic activity was measured using the fluorogenic substrate Na-benzyloxycarbonyl-L-glycyl-prolylarginine 7-amino-4-methylcoumarin (Z-GPR-AMC) in lmM Tris, pH 8.0, containing 1ram CaCI2, lOmM NaCI with and without CuCI2. To minimize the interactions between Tris buffer and copper ions, a low concentration of Tris (1 raM) was used in the assays. 103
TIBS 17 - MARCH 1992 Table II. Partial amino acid sequence alignment: of trypsin and tonin 91
92
93
94
95
A
Trypsin
H
P
N
F
D
.
Tonin
H
P
D
Y
I
P
B
C .
L
D .
I
E .
V
F .
T
N
G
H .
D
I
J
K
. T
E
Q
P
96
97
98
99
i00
i01
102
R
K
T
L
N
N
D
V
H
D
H
S
N
D
aThe amino acid sequences of trypsin and tonin from position 91 to 102 (chymotrypsin numbering system) were arranged by aligning the catalytic Asp at position 102 and the highly conserved His at position 91.
The biological significance of these protein engineering studies on trypsin can be seen in the metal inhibition observed in the enzyme tonin where metalloregulation of proteases reaches its zenith. Rat tonin is a trypsin-like Set protease whose physiological role is not known. An amino acid sequence alignment of tonin and trypsin from position 91 to position 102 (Table II) shows that, relative to trypsin, rat tonin has an 11 amino acid insertion between amino acid residues 95 and 96. The three-dimensional structure of tonin in the presence of zinc shows that these 11 amino acids form a flexible loop adjacent to His57. This alignment also identifies two His amino acid residues at positions 97 and 99. Both His97 and 99 of tonin, along with His57 coordinate a single zinc ion4L This coordination complex repositions the imidazole of His57 out of catalytic register and accounts for the inactivation of tonin in the presence of a transition metal ion. An observed K~ for the inhibition of tonin with copper was determined to be 680 nM (J. N. Higaki, unpublished), demonstrating that tonin binds copper better than trypsinR96H (K~=21BM). This tight binding is expected since the coordination of a metal ion by tonin involves three His side chains4L The transition metal ion bound to trypsinR96H is coordinated via two proximal His side chains, one of which is His57. Although the binding is strong enough to inactivate trypsinR96H, based on the structure of rat tonin, the binding of the metal ion might be enhanced by engineering three imidazole side chains for metal coordination instead of two.
Significance and potential applications Other proteins are being designed to of engineered metalloregulation in enzymes. test the general applicability
In one example, a metal-dependent regulatory site involving a specific His substitution was also engineered into subtilisin, a structurally distinct class of serine proteases with a wide commercial interest. In this enzyme, the substituted His was placed adjacent to the catalytic
104
His64, resulting in a subtilisin variant that was sensitive to exogenous copper (C. Paech, unpublished). Similarly, a metal-dependent regulatory site involving substituted His amino acid residues was recently engineered into glycogen phosphorylase. In this example, the binding of a Zn2÷ion bridges the two subunits of this dimeric protein and, in so doing, induces a conformational change that results in the allosteric activation of the enzyme (M. Browner, unpublished). The ability to engineer a metal binding site into an enzyme to regulate its activity may eventually find important in vitro applications. In the case of Ser proteases, the ability to inhibit protease activity during the purification of the enzyme would eliminate problems associated with the proteolytic degradation of the protein. Precise control of enzyme activity would make a variety of enzymes useful as biochemical reagents. By establishing the ability to engineer specific metal binding sites into biological macromolecules, one can begin to construct genetically altered organisms that can be used for purposes such as microbial marking and bioremediation. Metal regulation of enzyme activity might also find significant applications in vivo, thereby setting the stage for creating enzymes and proteins of great therapeutic value.
Acknowledgements We are grateful for the stimulating discussions we have had with Dr B. Haymore and for a preprint of an unpublished review of metal binding sites in proteins by Dr J. Tainer. This work was supported by NSF grants DMB-8904956 and EET-8807179 (CSC) and NIH grant DK-39304 (RJF). J. N. H. was an NIH postdoctoral fellow supported by fellowship GM-11598.
References 1 Ibers, J. A. and Holm, R. H. (1980) Science 209, 223-235 2 Robson, B. and Gamier, J. (1988) in Introduction to Proteins and Protein Engineering, pp. 195-224, Elsevier 3 Berg, J. M. (1987) Cold Spring Harbor Syrup. Quant. Biol. 52, 579-585 4 Vallee, B. L. and Auld, D. S. (1990)
Biochemistry 29, 5649-5659 5 Cunningham, B. C., Bass, S., Fuh, G. and Wells, J. A. (1991) Science 250, 1709-1712 6 Zhang, Z-Y. et al. (1991) Biochemistry 30, 8717-8721 7 Karlstrom, A. R. and Levine, R. L. (1991) Proc. Natl Acad. SCi. USA 88, 5552-5556 8 Snyder, E. E., Buoscio, B. W. and Falke, J. J. (1990) Biochemistry 29, 3937-3943 9 Sulkowski, E. (1985) Trends Biotechnol. 3, 1-7 10 Arnold, F. H. (1990) 8iotechnology 9, 151-156 11 Sundberg and Martin (1974) Chem. Rev. 74, 471-517 12 Yamashita, M. M., Wesson, M., Eisenman, G. and Eisenberg, D. (1990) Proc. Natl Acad. Sci. USA 87, 5648-5652 13 Chakrabarti, P. (1991) Protein Eng. 4, 57-63 14 Suh, S-S., Haymore, B. L. and Arnold, F. H. (1991) Protein Eng. 4, 301-305 15 Tainer, J. A., Roberts, V. A. and Getzoff, E. D. (1991) Curr. Opin. Biotechnol. 2, 582-591 16 Hochuli, E. et al. (1988) Biotechnologff 6, 1321-1325 17 Smith, M, Furman, T. C., Ingolia, T. D. and Pidgeon, C. (1988) J. Biol. Chem. 263, 7211-7215 18 Arnold, F. H. and Haymore, B. L. (1991) Science 252, 1796-1797 19 Lawson, D. M. et al. (1991) Nature 349, 541-544 20 Hatfull, G. F. et al. (1989) J. Mol. Biol. 208, 661-667 21 Iverson, B. L. et al. (1990) Science 249, 659~62 22 Cochran, A. G. and Schultz, P. G. (1990) Science 249, 781-783 23 Higaki, J. N. et al. (1990) Biochemistry29, 8582-8586 24 Fersht, A. R. (1985) Enzyme Structure and Mechanism, W. H. Freeman 25 Matsumura, M. and Matthews, B. W. (1989) Science 243, 792-794 26 Grabarek, Z. et al. (1990) Nature 345, 132-135 27 Fujimori, K. et al. (1990) Nature 345, 182-184 28 Russell, A. J. and Fersht, A. R. (1987) Nature 328, 496-500 29 McGrath, M. E. et al. Biochemistry(in press) 30 Corey, D. R. and Schultz, P. G. (1989) J. Biol. Chem. 264, 3666-3669 31 Drey, C. N. C. and Fruton, J. S. (1965) Biochemistry4, 1258-1263 32 Hemdan, E. S., Zhao, Y-J., Sulkowski, E. and Porath, J. (1989) Proc. Natl Acad. Sci. USA 86, 1811-1815 33 Ponder, J. W. and Richards, F. M. (1987) J. Mol. Biol. 193, 775-791 34 Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358 35 Chambers, J. L. et al. (1974) Biochem. Biophys. Res. Commun. 59, 70-74 36 Brothers, H. M. and Kostic, N. M. (1990) Biochemistry 29, 7468-7474 37 Young, M. and Koroly, M. J. (1980) Biochemistry 19, 5316-5321 38 Rees, D. C., Lewis, M. and Lipscomb, W. N. (1983) J. Mol. Biol. 168, 367-387 39 Matthews, B. W. et al. (1972) Nature 238, 37-41 40 Christianson, D. W. and Alexander, R. S. (1989) J. Am. Chem. Soc. 111, 6412-6419 41 Fujinaga, M. and James, M. N. G. (1987) J. Mol. Biol. 195, 373-396
REVIEWS A RIGOROUS ~ S T of our understanding of structure-activity relationships in proteins is the use of those relationships in altering the function of a protein in a prescribed fashion. The field of protein engineering has progressed to the point where the rational design of proteins is now providing an avenue for the development of proteins as reagents with a practical use. This is especially true of proteins whose activities and/or structures are dependent upon coordinating metal ions. Our knowledge of such metalloproteins has expanded, allowing basic rules governing protein-metal interactions to be formulated and applied in engineering metalloproteins for specific purposes. A variety of metal ions are known to interact with a large number of proteins required for a host of physiological processes L2. The role of the bound metal ion in these metalloproteins is varied but, in all cases, metal ions possess features that make them useful as cofactors in proteins 3. Metal ions associated with proteins have been implicated in substrate binding, catalysis, protein folding and stabilization. Metal ions play important roles in mediating protein-protein and protein-DNA intermolecular interactions4,s and have also been implicated as physiological regulators of enzymatic activity6,7. Fundamental principles based on protein-metal interactions of naturally occurring metalloproteins can now be put to use in creating metalloproteins with specific characteristics. Of particular interest is the design of metal iondependent regulatory sites in enzymes, since the ability to control the activity of an enzyme by manipulating the availability of metal ions in the external media may have useful clinical or industrial applications.
Chemical principlesof metal bindingsites A survey of metalloproteins of known three-dimensional structure has identJ. N. Higaki and C. S. Craik are at the " Department of PharmaceuticalChemistryand R. J. Flettorick and C. S. Craik are at the Department of Biochemistryand Biophysics, Universityof California, San Francisco, CA 94143, USA.
100
Engineered metalloregulation in enzymes
Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.
ified important features of metal-protein with metal ions, the infrequent occurinteractions that can be used in the rence of a free sulfhydryl group in prodesign of a metal-dependent regulatory teins makes His the primary amino acid siteL Since it is the unique electronic residue involved in metal binding ~°," properties (charge, coordination number and the one most commonly used in and geometry) of certain metal ions engineering metal binding sites. that make them useful in biological sysA method for the prediction of metal tems 2,8,one of the first considerations is binding sites in proteins has been prothe type of metal ion to be used. Among posed 12 that describes important structhe most common metal ions in naturally tural features of a protein environment occurring metalloproteins are first row that favor metal binding. However, the transition metal ions (Zn 2+, Cul+/2+, orientation of the amino acid residues Fe2÷/3+) with typical coordination num- serving as ligands must also compbers ranging from 4 to 6. These ions lement the preferred coordination gehave favorable coordination geometries ometry of the bound metal ion 2. In a that enable them to form stable interac- number of metalloproteins, the presence tions with the amino acid side chains of of His side chains that are able to adopt proteins. Although the stability of preferred orientations provides clues as metal-ligand interactions is determined to how adjacent imidazole side chains in a large part by features of the metal can form stable metal binding sites ~3. ion itself, it is also dependent on both When two or more His side chains the availability and the conformation of are available [o coordinate a metal ion, associated protein ligands. certain unique spatial orientations of Amino acid residues that function as these side chains can favor enthalpic good metal ligands are typically those and entropic effects leading to tighter that contain electron-donating atoms binding (chelation) of a metal ion9. For (S, O or N) 9. Although this group example, a His-X-X-X-His motif in an includes those containing unionizahle m-helix of a number of naturally-occurgroups (Trp, Ser, Met) the strongest ring proteins possesses two correctly interactions typically involve amino positioned imidazoles that together can acid side chains with ionizable groups chelate a metal ion. Such a structure (Asp, Glu, Cys, His). Since it is the can be engineered into proteins to engreater basicity of the functional group hance their meta! binding properties ~4. (higher pKa) that makes it the better By using these and other principles of ligand, the order of copper binding metal binding derived from both theorability for these amino acids is etical work and empirical observations Cys > His > Asp/GIu. Basic amino acid on metalloproteins of known strucresidues Lys and Arg rarely participate tures, strong metal binding sites are in metal binding and despite the fact now being engineered into proteins for that Cys forms such strong interactions a variety of purposes. © 1992,ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00
TIBS 17 - MARCH 1992
Within the past few years, interest in the design of metal binding sites in proteins has been expanding rapidly as more applications of engineered metal binding sites develop, many of which have recently been reviewed ~s. For example, metal binding sites have been introduced into proteins to stabilize them against denaturation or proteolytic degradation. They have also been engineered into proteins to assist in their purification ~5-~8, crystallization ~9, and X-ray crystal structure determination 2°. lverson et al. 2~ have reported the design of a metalloantibody that contains a His coordination site for metals in the antigen binding pocket. This is an important step forward in the design of antibodies that could catalyse redox and hydrolytic reactions. An exciting development in catalytic antibodies was recently described that involves the isolation and initial characterization of an antibody capable of catalysing porphyrin metallation 22. Further analysis of enzymes generated in this fashion should contribute unique and insightful information to the database of metalprotein interactions.
lated more precisely. In addition to enhancing the zd + control one has over the enzymatic reaction, this 2.1 2.1 mechanism of enzyme ~.~ .~ 102° %.. /"'~NE' 'Ne7 \ regulation also permits a unique analysis of structure-activity relationships in the engineered protein. Several proteins have been engineered so that their activity can be regulated in vitro by exploiting disulfide bonds 25,26,salt bridges 27 and electrostatics 28,~9. Based on the database of information regarding Figure 1 metalloprotein structure An ideal metal binding site. The side chains of His142 and His146, along with Glu166 (not shown) comprise and on the natural role of the zinc binding site of thermolysin (file 2TLN, Brookmetal ions in regulating enhaven Protein Data Bank). This naturally-occurring zyme activity, metal bindmetal binding site illustrates the orientation of the His ing sites are presently side chains required for strong metal binding. The imidabeing engineered into enzole side chains of His142 and His146 are positioned zymes for enhanced regu102" relative to each other and are situated 2.1 A from the bound zinc ion. The corresponding Ca.of these two lation. One of the first examino acid residues are less than 13 A apart, This amples of an engineered metal binding geometry was used as a template for regulatory site involving locating potential metal binding sites in proteins of the binding of a metal ion known three-dimensional structure. was that reported for staphylococcal nuclease 3°. Metal.dependent enzyme regulation The ability of sulfhydryl groups to co- near the enzyme active site could result A novel application of an engineered ordinate metal ions was exploited in in a local conformational change upon metal binding site in proteins is the redesigning staphylococcal nuclease so binding the metal ion that would alter introduction of such a site into a pro- that a free cysteine was exposed at the the catalytic properties of the enzyme. tease for regulation of enzyme activityz3. base of the substrate binding pocket. Co- By making the metal binding site depenThis mechanism of metalloregulation ordination of this sulfhydryl group with dent upon an active site amino acid, relies on the binding of metal ions to either organic mercurial compounds or enzyme activity provides a rapid evaluinduce local structural changes in an copper occluded the substrate binding ation of the original design principles. enzyme that result in altered catalytic pocket rendering the enzyme inactive. The three-dimensional structure of properties. Creation of such a metal By removing the bound metal ion with an enzyme can be searched for suitable switch is based on the three-dimensional strong chelating agents, the pocket was sites that, when substituted with His structure of the enzyme, the chemical cleared and the enzyme activity was amino acid residues, would form a mechanism and many of the concepts restored 3°. Although powerful, this strong metal binding site capable of generally accepted regarding metal ion method may not always be possible in altering the catalytic activity of an encoordination in proteins. The informa- proteins that contain naturally occur- zyme. The unique ability of an ention gained from these experiments may ring cysteines or disulfide bonds since gineered metal binding site to function lead to a general method for engineering the presence of a free sulfhydryl might in regulation relies on the ability of the a metal regulatory site into any enzyme. interfere with protein folding in certain bound metal ion to alter the conforThe regulation of enzyme activity is expression systems. Futhermore, the mation of amino acid residues critical an~essential feature of all physiological oxidation state of cysteine may be dif- for activity. If sites for His substitutions processes. In vitro, the activity of an en- ficult to control in the presence of are chosen that would provide a potenzyme can be regulated by manipulating metal ions, making histidines an attrac- tial metal binding site involving the environmental factors such as pH, tem- tive alternative to cysteines for en- active site His, the coordination of this perature, ionic strength, substrate con- gineered metal chelation. critical His to a bound metal ion would centration, and exposure to specific reversibly inactivate the enzyme by inhibitors. Manipulating the in vitro Histidines as targets for engineered preventing its participation in catalysis. activity of an enzyme has led to a metal.dependent regulation The potential amino acid pairs of the wealth of knowledge concerning the The approach taken in introducing a metal switch need to satisfy five importrole of enzymes in biochemical reac- metal-dependent regulatory site into an ant criteria based on our current undertions and the mechanisms employed to enzyme is based on the ability of imida- standing of metal binding sites: carry out their specific functions 24. zoles 3~ and of properly oriented His With the advent of recombinant DNA amino acid side chains 9,32 to chelate (1) The side chains should be solvent technology came the ability to engineer transition metal ions. Strategic placeaccessible for exogenous metal the enzyme itself so that it can be regu- ment of a histidine amino acid residue binding.
/
101
TIBS 17 - MARCH 1992
(2) The distances between the mcarbons of the unsubstituted side chains should be less than 13.0 ]~ to allow the corresponding imidazole side chains to chelate the metal. (3) The His side chains should be any one of five favorable rotational conformers 33 and still coordinate the metal ion. If the torsional angles of the side chains correspond to an unfavorable free energy, then some free energy gain that derives from chelating the metal will be lost. However, the metal sites in natural proteins are often coordinated to His side chains which are not favorable rotamers suggesting that this is not a stringent requirement. (4) All atoms of the substituted His side chains should be further than 3.0 A from other atoms of the pro-
(5)
rein unless a hydrogen bond could be formed. This design criteria would prevent any steric overlap that might cause distortions in structure. Binding of the metal ion should induce a conformational change that could alter the catalytic properties of the enzyme.
Computer programs that employ these or similar criteria are available or are currently being developed (Biosym Technology). A quintessential example of a metal binding site is found in thermolysin (file 2TLN, Brookhaven Protein Data Bank; Fig. 1). Two histidines help coordinate the Zn 2÷ in this metalloprotease. The cis orientation of the histidines liganded to the Zn 2÷ places them at an angle of approximately 102°. The
Figure 2 Schematic representation of the metal switch in trypsinR96H. The Ca positions of the active site region are shown along with the modeled coordination complex formed between a single Cu2+ ion, His96 and His57 of trypsinR96H. The coordinates for the side chain of Arg96 were replaced with the coordinates for a His and the torsional angles of His57 and His96 were adjusted manually to superimpose with His142 and His146 of thermolysin. His142 and His146, along with Glu172, bind the Zn 2+ ion in the active site of thermolysin and represent a functional coordination complex. After adjustment of the X2 angle for the His57 side chain by 50 °, a root mean square superposition of less than 0.4/~ was achieved for the His atomic coordinates of thermolysin and trypsinR96H. This coordination complex places the Cu2÷ ion at a position that is simultaneously 2.2 A (subtending an angle of 95") from the N¢ nitrogen atom of both His side chains. Subroutines from the program Insight (Biosym Technologies, San Diego) were used to perform the alignments and torsional angle adjustments of the side chains. His57 is normally positioned in the active site of trypsin where it is within hydrogen bonding distance to the side chains of Aspl02 and Ser195. These three residues cor~stitute the functional catalytic triad of trypsin. The N¢ atom of the His57 side chain is expected to move 6 A (curved arrow) on coordination with a Cu2÷ ion that binds to His96. The arrows pointing from the N~ atoms of the two imidazole groups to the Cu2÷ ion represent coordinate covalent bonds. Removal of the bound Cu2÷ ion allows the His57 side chain to rotate back into the active site thereby reestablishing the functional catalytic triad. A space-filling version of this figure is shown on the front cover of this issue.
102
distance between the metal and the coordinated nitrogen of the imidazole is approximately 2 A. This configuration exemplifies the His-metal-His template that was used to search the threedimensional structure of a protein for potential metal binding sites.
Metal-dependent regulation of trypsin Although the criteria described above could be met in various proteins, trypsin was a particularly attractive enzyme to test the design principles. Trypsin catalyses the hydrolysis of ester and amide bonds carboxy-terminal to the c(-carbon of Arg and Lys amino acid residues. It is a member of a large and well-studied family of serine (Set) proteases that all have a nucleophilic Set amino acid residue at the active site. In the catalytic mechanism of Ser proteases, His57 serves initially as a general base to increase the nucleophilicity of the essential Ser and subsequently as a general acid to donate a proton to the leaving group 24,34. Because it is critical for catalytic activity, repositioning of the His57 side chain away from the active site will disrupt the conformation of the catalytic triad, rendering the enzyme inactive. To reposition the imidazole of His57, a second His was placed in the vicinity of His57, thereby forming a two-histidine metal binding site 23. Based on the five criteria listed above and using the coordinates for rat trypsin (file 2RTM, Brookhaven Protein Data Bank), nine pairs of potential His pairs satisfy the first four criteria set by the search algorithm; however, only His57 together with a His substituted for Arg96 satisfy the fifth criteria. Computer modeling studies indicated that, in the presence of Cu2+, the side chain of His57 can rotate 90 ° ab6ut the C~-Cp bond forming a His96-His57-Cu 2+ coordination complex that can be superimposed onto the natural metal-chelating site of thermolysin. The resultant metal switch is shown in Fig. 2; His57 is shown both in catalytic register within the catalytic triad, and out of catalytic register when coordinated to a single Cu2÷ion. Having identified a potential site (position 96) for His substitution, sitedirected mutagenesis of rat anionic trypsin was used to substitute Arg96 with a His (trypsinR96H) 23. The velocity of the His96-modified enzyme could be altered by the presence of metal ions as dramatically demonstrated in Fig. 3. The kinetic parameters of trypsinR96H in comparison to trypsin (Table I) also
TIBS 1 7 - MARCH 1992
show that the enzyme closely resembles trypsin in the absence of Cu2÷, but has a compromised kca t value in the presence of 0.4 mM Cu2÷. Removal of the bound Cu2+ restores the variant to its active state. This is a textbook case of non-competitive inhibition with a K~ of 21 ~M for copper, 49 tim for nickel and 128 laM for zinc23. The order of binding is, as expected, based on the binding of those metal ions to imidazole. The Lewis acidity of the metal governs the order of binding to the ligand; however, the absolute value of the association constant is critically dependent upon the geometrical constraints of the metal binding site imposed by the protein scaffold. The three-dimensional structure of the engineered metal switch in the presence and absence of metal should provide a structural framework for analysing its affinity for transition metals. The structure will also be invaluable for creating subsequent metal binding sites with higher affinity and with greater selectivity. Serine/metallo-protease link?
(a)
J
B
C D
0 I
E
-~
,
I
~
I
I
~
I
(b) 60
-R,
40
20
- an evolutionary
Wild-type trypsin was shown to be partially and reversibly inhibited by binding of a single metal ion at very high concentrations of Cu2÷ (Ref. 23). This was consistent with the observation that the Asp102-His57 couple of trypsin is inhibited by binding of a silver ion35. Other serine proteases have also been reported to be inhibited by metal ions. A few notable examples are the inhibition of catalytic activity of et-chymotrypsin and (~-lytic protease3E The autocatalytic activation of the zymogen form of the ~-subunit of nerve growth factor has also been shown to be controlled by zinc 37. Although the mode of metal interaction has not been characterized for these serine proteases, it is possible that they all have a similar mechanism of metal binding. Thus, Asp~His couples that are capable of binding metal ions might naturally play a role in protein folding or in enzyme regulation. The binding of a metal ion at the Asp102-His57 couple in the catalytic triad of Ser proteases is structurally similar to the way in which metal ions are coordinated as a component of the catalytic triad of zinc metalloproteases such as carboxypeptidase A38 and thermolysin39. The metal binding Asp-His couple of serine proteases may be an evolutionary structural motif linking the active sites of serine proteases to zinc
[]
A
0"-
t
0
,
50
I
~
i 200
I
100
150
[ Z - G P R - A M C ] (pM) Figure 3 Velocity versus substrate concentrations for trypsinR96H (a) and trypsin (b) at various concentrations of Cu2÷. The activity of 1.25 nM enzyme was monitored fluorometrically at 25°C using varying concentrations of the tripeptide substrate, Z-GPR-AMC. Assay conditions were as follows: 1 mM Tris, pH 8.0 containing 1 mM CaCl2, 10 mM NaCI and (A) 0 mM CuCl2; (B) 0.02 mM CuCl2; (C) 0.04 mM CuCl2; (D) 0.1 mM CuCl2; (E) 0.2 mM CuCl2. The excitation wavelength was set at 380 nm and the emission wavelength was set at 460 nm. (a) The maximum velocity for trypsinR96H at high Z-GPR-AMC concentrations (200 IJM) reached an increasingly lower limit at higher concentrations of Cu2÷. (b) The activity of 1.25 nM wildtype trypsin is relatively unaffected by Cu2÷ ions using the same conditions described in (a).
metalloproteases 4°. In the serine proteases, the Asp-His couple might have recruited an activated serine, resulting in the catalytic triad. The metallopro-
teases, alternatively, could have used the Asp-His couple to incorporate a metal ion at the active site of the enzyme.
Table I. Kinetic parameters a of trypsin and trypsinR96H
Enzyme
Trypsin TrypsinR96H
CuCl2 (mM)
kcat (min-1)
Km (I.IM)
kcat / K m (minlpM-1)
(pM)
0.0 0.4
3345 2510
14.1 16.7
237.3 150.4
1 600
0.0 0.4
2464 495
7.5 26.0
331 19.0
21
K i
aproteolytic activity was measured using the fluorogenic substrate Na-benzyloxycarbonyl-L-glycyl-prolylarginine 7-amino-4-methylcoumarin (Z-GPR-AMC) in lmM Tris, pH 8.0, containing 1ram CaCI2, lOmM NaCI with and without CuCI2. To minimize the interactions between Tris buffer and copper ions, a low concentration of Tris (1 raM) was used in the assays. 103
TIBS 17 - MARCH 1992 Table II. Partial amino acid sequence alignment: of trypsin and tonin 91
92
93
94
95
A
Trypsin
H
P
N
F
D
.
Tonin
H
P
D
Y
I
P
B
C .
L
D .
I
E .
V
F .
T
N
G
H .
D
I
J
K
. T
E
Q
P
96
97
98
99
i00
i01
102
R
K
T
L
N
N
D
V
H
D
H
S
N
D
aThe amino acid sequences of trypsin and tonin from position 91 to 102 (chymotrypsin numbering system) were arranged by aligning the catalytic Asp at position 102 and the highly conserved His at position 91.
The biological significance of these protein engineering studies on trypsin can be seen in the metal inhibition observed in the enzyme tonin where metalloregulation of proteases reaches its zenith. Rat tonin is a trypsin-like Set protease whose physiological role is not known. An amino acid sequence alignment of tonin and trypsin from position 91 to position 102 (Table II) shows that, relative to trypsin, rat tonin has an 11 amino acid insertion between amino acid residues 95 and 96. The three-dimensional structure of tonin in the presence of zinc shows that these 11 amino acids form a flexible loop adjacent to His57. This alignment also identifies two His amino acid residues at positions 97 and 99. Both His97 and 99 of tonin, along with His57 coordinate a single zinc ion4L This coordination complex repositions the imidazole of His57 out of catalytic register and accounts for the inactivation of tonin in the presence of a transition metal ion. An observed K~ for the inhibition of tonin with copper was determined to be 680 nM (J. N. Higaki, unpublished), demonstrating that tonin binds copper better than trypsinR96H (K~=21BM). This tight binding is expected since the coordination of a metal ion by tonin involves three His side chains4L The transition metal ion bound to trypsinR96H is coordinated via two proximal His side chains, one of which is His57. Although the binding is strong enough to inactivate trypsinR96H, based on the structure of rat tonin, the binding of the metal ion might be enhanced by engineering three imidazole side chains for metal coordination instead of two.
Significance and potential applications Other proteins are being designed to of engineered metalloregulation in enzymes. test the general applicability
In one example, a metal-dependent regulatory site involving a specific His substitution was also engineered into subtilisin, a structurally distinct class of serine proteases with a wide commercial interest. In this enzyme, the substituted His was placed adjacent to the catalytic
104
His64, resulting in a subtilisin variant that was sensitive to exogenous copper (C. Paech, unpublished). Similarly, a metal-dependent regulatory site involving substituted His amino acid residues was recently engineered into glycogen phosphorylase. In this example, the binding of a Zn2÷ion bridges the two subunits of this dimeric protein and, in so doing, induces a conformational change that results in the allosteric activation of the enzyme (M. Browner, unpublished). The ability to engineer a metal binding site into an enzyme to regulate its activity may eventually find important in vitro applications. In the case of Ser proteases, the ability to inhibit protease activity during the purification of the enzyme would eliminate problems associated with the proteolytic degradation of the protein. Precise control of enzyme activity would make a variety of enzymes useful as biochemical reagents. By establishing the ability to engineer specific metal binding sites into biological macromolecules, one can begin to construct genetically altered organisms that can be used for purposes such as microbial marking and bioremediation. Metal regulation of enzyme activity might also find significant applications in vivo, thereby setting the stage for creating enzymes and proteins of great therapeutic value.
Acknowledgements We are grateful for the stimulating discussions we have had with Dr B. Haymore and for a preprint of an unpublished review of metal binding sites in proteins by Dr J. Tainer. This work was supported by NSF grants DMB-8904956 and EET-8807179 (CSC) and NIH grant DK-39304 (RJF). J. N. H. was an NIH postdoctoral fellow supported by fellowship GM-11598.
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Biochemistry 29, 5649-5659 5 Cunningham, B. C., Bass, S., Fuh, G. and Wells, J. A. (1991) Science 250, 1709-1712 6 Zhang, Z-Y. et al. (1991) Biochemistry 30, 8717-8721 7 Karlstrom, A. R. and Levine, R. L. (1991) Proc. Natl Acad. SCi. USA 88, 5552-5556 8 Snyder, E. E., Buoscio, B. W. and Falke, J. J. (1990) Biochemistry 29, 3937-3943 9 Sulkowski, E. (1985) Trends Biotechnol. 3, 1-7 10 Arnold, F. H. (1990) 8iotechnology 9, 151-156 11 Sundberg and Martin (1974) Chem. Rev. 74, 471-517 12 Yamashita, M. M., Wesson, M., Eisenman, G. and Eisenberg, D. (1990) Proc. Natl Acad. Sci. USA 87, 5648-5652 13 Chakrabarti, P. (1991) Protein Eng. 4, 57-63 14 Suh, S-S., Haymore, B. L. and Arnold, F. H. (1991) Protein Eng. 4, 301-305 15 Tainer, J. A., Roberts, V. A. and Getzoff, E. D. (1991) Curr. Opin. Biotechnol. 2, 582-591 16 Hochuli, E. et al. (1988) Biotechnologff 6, 1321-1325 17 Smith, M, Furman, T. C., Ingolia, T. D. and Pidgeon, C. (1988) J. Biol. Chem. 263, 7211-7215 18 Arnold, F. H. and Haymore, B. L. (1991) Science 252, 1796-1797 19 Lawson, D. M. et al. (1991) Nature 349, 541-544 20 Hatfull, G. F. et al. (1989) J. Mol. Biol. 208, 661-667 21 Iverson, B. L. et al. (1990) Science 249, 659~62 22 Cochran, A. G. and Schultz, P. G. (1990) Science 249, 781-783 23 Higaki, J. N. et al. (1990) Biochemistry29, 8582-8586 24 Fersht, A. R. (1985) Enzyme Structure and Mechanism, W. H. Freeman 25 Matsumura, M. and Matthews, B. W. (1989) Science 243, 792-794 26 Grabarek, Z. et al. (1990) Nature 345, 132-135 27 Fujimori, K. et al. (1990) Nature 345, 182-184 28 Russell, A. J. and Fersht, A. R. (1987) Nature 328, 496-500 29 McGrath, M. E. et al. Biochemistry(in press) 30 Corey, D. R. and Schultz, P. G. (1989) J. Biol. Chem. 264, 3666-3669 31 Drey, C. N. C. and Fruton, J. S. (1965) Biochemistry4, 1258-1263 32 Hemdan, E. S., Zhao, Y-J., Sulkowski, E. and Porath, J. (1989) Proc. Natl Acad. Sci. USA 86, 1811-1815 33 Ponder, J. W. and Richards, F. M. (1987) J. Mol. Biol. 193, 775-791 34 Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358 35 Chambers, J. L. et al. (1974) Biochem. Biophys. Res. Commun. 59, 70-74 36 Brothers, H. M. and Kostic, N. M. (1990) Biochemistry 29, 7468-7474 37 Young, M. and Koroly, M. J. (1980) Biochemistry 19, 5316-5321 38 Rees, D. C., Lewis, M. and Lipscomb, W. N. (1983) J. Mol. Biol. 168, 367-387 39 Matthews, B. W. et al. (1972) Nature 238, 37-41 40 Christianson, D. W. and Alexander, R. S. (1989) J. Am. Chem. Soc. 111, 6412-6419 41 Fujinaga, M. and James, M. N. G. (1987) J. Mol. Biol. 195, 373-396
