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Table 3

Residues in the active sites of the enzymes studied

Enolase (1ebg)

Residue

Role

R374

Interacts with C2 phosphate oxygen of 2-phosphoglycerate (2-PGA). May be important in altering the pKa of K345 in order to allow it to be a good catalytic base [Sup1].

S375

Interacts with C2 phosphate oxygen of 2-PGA [Sup1].

H159

Interacts with C2 phosphate oxygen of 2-PGA [Sup1]. Within a loop involved in a conformational change upon substrate binding [Sup2]. Previously proposed as the catalytic base [Sup3], however, H159A mutant still retained .2% of native activity [Sup4].

S39

Binds lower affinity Mg+2 ion. [Sup5]

Q167

Interacts with C1 carboxylate oxygen of 2-PGA [Sup6].

H373

Interacts with C3 hydroxyl oxygen of 2-PGA [Sup1]. Brewer et. al. states that H373 involves hydrogen bonding with E168 and E211, rather than acting as the catalytic acid. [Sup7]

E168

May be essential for proper orientation of K396 and E211, as well as pKa adjustment of these residues [Sup8].

E211

Catalytic acid which donates proton to C3- hydroxyl group of 2-PGA and causes H2O to leave substrate [Sup8].

K345

Catalytic base; abstracts proton from C2 [Sup8].

A38

Interacts with C2-phosphate oxygen of 2-PGA [Sup1]

G37

Hinge residue of active site loop (G37-H43) [Sup8]

D320

Binds to higher affinity Mg+2 ion [Sup8]

K396

Interacts with one of the C1 carboxylate oxygens to stabilize the redistribution of negative charge formed in the intermediate [Sup8].

D246

Binds to higher affinity Mg+2 ion [Sup8].

E295

Binds to higher affinity Mg+2 ion [Sup8].
Ribonuclease T1 (1rnt)

Residue

Role

Y45

Backbone hydrogen bonds to O6 of guanidine moiety. Lid of the guanine binding site [Sup9].

F100

May enhance electrostatic interactions between substrate and active site via. a change in the dielectric constant[Sup9].

E58

Catalytic base. Abstracts proton from 2'-hydroxyl oxygen on ribose [Sup9].

Y42

Side chain is involved in aromatic stacking interaction with guanine base [Sup9].

H40

Works cooperatively with E58 in abstraction of proton from 2'-hydroxyl oxygen on ribose [Sup9].

N43

Backbone NH interacts with guanine N7 [Sup10].

P73

Involved in surface loop (G71-G74), which affects the specificity of the 2' nucleotide [Sup11].

E46

Side chain O?1 hydrogen bonds with guanidine N1H; Side chain O?2 hydrogen bonds with guanidine N2H1 [Sup10].

N98

Confers specificity for cytidine in the leaving group [Sup9]. Backbone oxygen hydrogen bonds to guanidine N2H2 (exocyclic amine) [Sup10].

Y38

Side chain hydrogen bonds to one of the phosphate oxygens [Sup9].

N36

Interacts with the ribose moiety of the leaving nucleoside [Sup9].

H92

Catalytic acid; protonates the leaving group[Sup9].

R77

May neutralize the 2nd negative charge that develops on one of the phosphate oxygens [Sup9].

N44

Backbone NH hydrogen bonds with guanidine O6 [Sup10].

K41

Bordering residue to guanidine binding segment (Y42-E46) [Sup10].
Triosephosphate isomerase (2ypi)

Residue

Role

I170

Part of loop 6 (which includes catalytic E165). [Sup12, Sup13]

V231

Part of 'loop 8' [actually a 31o helix]. (NOT involved in a conformational change, unlike loop 7). Forms the base of the binding pocket where the phosphate moiety will bind. [Sup13]

L230

See note on V231 [Sup13]

G210

Part of loop 7. Involved in a conformational change. When closed, makes hydrogen bonds between the backbone and the substrate's phosphate oxygens. [Sup13]

E165

Catalytic base which abstracts the Pro-R proton from C1 of DHAP. Part of loop 6 [Sup14]

G232

See note on L230. Hydrogen bonds to phosphate oxygen. [Sup13]

S211

See note on G210. [Sup13]

G209

Part of loop 7. Involved in a conformational change. [Sup13]

A212

Part of loop 7. Involved in a conformational change. [Sup13]

K12

Catalytic residue which assists in providing positive charge necessary for substrate binding by interacting with the C2 oxygen of the substrate. [Sup14, 15]

H95

Catalytic residue responsible for shuttling protons between the two oxygens (connected to C1 and C2, respectively) in the intermediate [Sup16]

G233

See note on G232. [Sup13]

N10

Considered to be part of the catalytic site. May work in conjunction with K12 and E165 to create an environment polar enough to facilitate proton transfer. Interacts with C1-oxygen of substrate. [Sup17]

G171

Hydrogen bonds to phosphate oxygen. [Sup12]
Trypsin (1tng)

Residue

Role

S190

S1 binding site (i.e. binding site for P1 residue) [Sup18]; hydrogen bonds with P1 side chain [Sup19]

W215

S1 binding site [Sup18]; contacts the backbone of the P3 residue [Sup19]

C191

S1 binding site [Sup18]; backbone forms van der Waals contacts with P1 residue (observed in PDB structure-1GDU), involved in disulfide bridge with C220 [Sup20]

S214

S1 binding site [Sup18]; backbone hydrogen bonds with the backbone N of the P1 residue [Sup19]

G216

S1 binding site [Sup18]; substrate specificity determining residue (by intersecting and lining the S1 site) [Sup18]

V213

Forms van der Waals interactions with the P1 side chains (observed in PDB structure-1GDU)

D189

S1 binding site [Sup18]; substrate specificity determining residue (supplies negative charge to interact with P1 residue) [Sup18]

C220

S1 binding site [Sup18]; backbone forms van der Waals contacts with P1 residue (observed in PDB structure-1GDU), involved in disulfide bridge with C191 [Sup20]

G219

S1 binding site [Sup18]; hydrogen bonds with P1 side chain [Sup21]

G226

S1 binding site [Sup18]; contributes to substrate specificity [Sup22]

S195

Catalytic nucleophile (part of catalytic triad) which attacks backbone carbonyl group [Sup23]; S1 binding site [Sup18]

Y228

S1 binding site [Sup18], outlines the periphery of the S1 site (observed in PDB structure-1GDU)

V227

S1 binding site [Sup18], outlines the periphery of the S1 site (and backbone hydrogen bonds with S214-W215) (observed in PDB structure-1GDU)

Q192

S1 binding site [Sup18]; side chain hydrogen bonds with the backbone O of the P2 residue [Sup19]

H57

Abstracts proton from S95 (part of catalytic triad) [Sup23]
Fructose-1,6-bisphosphatase (1fbc)

Residue

Role

D121

Coordinates with metal (Mg+2, Zn+2, K+ for example) [Sup24]; participates in a loop (D121-N125) which has a 1A conformational change occurring [Sup25]; contacts the sugar ring [Sup25].

M248

Backbone contacts the sugar ring [Sup24, Sup25].

K274

Hydrogen bonds to 6-phosphate of F2,6 bisphosphate inhibitor [Sup25]; Contacts the sugar ring [Sup25].

S247

Backbone contacts the sugar ring [Sup24].

G122

Hydrogen bonds to 2-phosphate of F2,6BP inhibitor [Sup25]; participates in a loop (D121-N125) which has a 1A conformational change occurring [Sup25].

L275

Makes van der Waals interactions with substrate. [Sup26]

G246

No explicit role stated. Makes van der Waals interactions with substrate in majority of Fructose-1,6-bisphosphatase structures .

E97

Coordinates with metal [Sup24]

Y244

Hydrogen bonds to 6-phosphate of F2,6BP [Sup25].

K264

See note for Y244 [Sup25]

N212

See note for Y244 [Sup25]

Y215

See note for Y244 [Sup25]

R243

Hydrogen bonds to 6-phosphate of F2,6BP (of adjacent monomer) [Sup25].

D74

Proposed to be catalytic base (working synergistically with E98). Abstracts protons from metal-hydroxide complex [Sup27].

E98

See note for D74 [Sup27]

D118

Coordinates with metal [Sup24].

E280

Contacts 'site 3' metal atom (NOTE: The 1-phosphate group also contacts the 'site 3' metal) [Sup28]

S123

See note for G122 [Sup25].

S124

See note for G122 [Sup25].

N125

See note for G122 [Sup25].

R276

See note for E280 [Sup28]

Haloalkane dehalogenase (2dhc)

Residue

Role

E56

Within 4 Å distance to the bromide ion [Sup31]

D124

Part of catalytic triad [Sup29], carrying out the nucleophylic attack on the halogene-bound C1 atom [Sup31]

W125

The NH group of the side chain stabilizes the Cla in the Michaelis complex [Sup31]

F128

May interact with the the Clb of the substrate in the Michaelis complex [sup29]

F164

May interact with the the Clbof the substrate in the Michaelis complex [sup29]

W175

The NH group of the side chain stabilizes the Cla in the Michaelis complex [Sup31]

F222

Within 4 Å distance to the bromide ion [Sup31]

P223

Within 4 Å distance to the bromide ion [Sup31]

V226

Within 4 Å distance to the bromide ion [Sup31]

D260

3rd member of the catalytic triad [Sup31]

L262

Blocks the tunnel that leads to the binding site [Sup31]

H289

General base [sup30]
Thermolysin (2tlx)

Residue

Role

N111

Backbone forms hydrogen bond with the leaving group [Sup32]

N112

Forms hydrogen bond with the leaving group [Sup32]

A113

Backbone forms hydrogen bond with the leaving group [Sup32]

F114

Contributes to the hydrophobic part of the S1' pocket

W115

Backbone forms hydrogen bonds with the substrate

F130

Contributes to the hydrophobic part of the S1' pocket

L133

Contributes to the hydrophobic part of the S1' pocket

V139

Contributes to the hydrophobic part of the S1' pocket

H142

Coordinates the Zn2+ ion in the active site [Sup32]

E143

General base [Sup32]

H146

Coordinates the Zn2+ ion in the active site [Sup32]

Y157

Contributes to the stabilization of the transition state [Sup32]

E166

Coordinates the Zn2+ ion in the active site [Sup32]

L202

Contributes to the hydrophobic part of the S1' pocket

R203

Forms hydrogen bonds with the carbonyl group of the substrate residue at P1' position; crucial for binding [Sup33]

H231

Contributes to the stabilization of the transition state [Sup32]

References

Sup1. Zhang, E., Brewer, J.M., Minor, W., Carreira, L.A., Lebioda, L. Mechanism of Enolase: The Crystal Structure of Asymmetric Dimer Enolase - 2-Phospho-D-glycerate/Enolase - Phosphoenolpyruvate at 2.0 A resolution. Biochemistry 36: 12526-12534 (1997)

Sup2. Reed, G.H. et. al. Structural and Mechanistic Studies of Enolase. Current Opinion in Structural Biology 6: 736-743 (1996)

Sup3. Vinarov, D. A., Nowak, T. Role of His159 in yeast enolase catalysis. Biochemistry, 18: 12138-12149 (1999)

Sup4. Brewer, J. M., Holland, M. J. Lebioda, L. The H159A mutant of yeast enolase 1 has significant activity. Biochem. Biophys.Res. Comm. 276: 1199-1202 (2000)

Sup5. Brewer, J. M., Glover, C. V. C., Holland, M. J. Lebioda, L. Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism. Biochim. Biophys. Acta. 1383: 351-355 (1998)

Sup6. http://www.ccs.uky.edu/accessgrid/newsite/WeitaoYang.pdf

Sup7. Brewer, J.M., Glover, C. V. C., Holland, M. J. Lebioda, L. Effect of site-directed mutagenesis of His 373 of yeast enolase on some of its physical and enzymatic properties. Biochim. Biophys. Acta. 1340: 88-96 (1997)

Sup8. Larsen, T. M., Wedekind, J. E., Rayment, I., Reed, G. H. A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 A resolution. Biochemistry, 35: 4349-4358 (1996)

Sup9. Steyaert, J. A Decade of Protein Engineering on Ribonuclease T1. Atomic Dissection of the Enzyme-Substrate Interactions. Eur. J. Biochem., 247: 1-11 (1997)

Sup10. Hubner, B., Haensler, M., and Hahn,U. Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment. Biochemistry, 38: 1371-1376 (1999)

Sup11. Balaji, P.V., Saenger, W., and Rao, V.S.R. Computer Modeling Studies on the Binding of 2',5'-linked Dinucleotide Phosphates to Ribonucleotide T1 - Influence of Subsite Interactions on the Substrate Specificity. Journal of Biomolecular Structure and Dynamics 10: 891-903 (1993)

Sup12. Joseph, D., Petsko, G.A., Karplus, M. Anatomy of a Conformational Change: Hinged "Lid" Motion of the Triosephosphate Isomerase Loop. Science 249: 1425-1428 (1990)

Sup13. Norledge, B.V., et. al. Modeling, Mutagenesis, and Structural Studies on the Fully Conserved Phosphate-Binding Loop (Loop 8) of Triosephosphate Isomerase: Toward a New Substrate Specificity. Proteins: Structure, Function and Genetics 42: 383-389 (2001)

Sup14. Alber, T.C., et. al. Crystallography and Site Directed Mutagenesis of Yeast Triosephosphate Isomerase: What Can We Learn about Catalysis from a "Simple" Enzyme? Cold Spring Harbor Symposia on Quantitative Biology 52: 603-613 (1987)

Sup15. Harris, T.K., Abeygunawardana, C., and Mildvan, A.S. NMR Studies of the role of Hydrogen Bonding in the Mechanism of Triosephosphate Isomerase. Biochemistry 36: 14661-14675 (1997)

Sup16. Zhang, Z., et. al. The Role of Water in the Catalytic Efficiency of Triosephosphate Isomerase. Biochemistry 38: 4389-4397 (1999)

Sup17. Wierenga, R.K., Borchert, T.V., Noble, M.E.M. Crystallographic Binding Studies with Triosephosphate Isomerases: conformational changes induced by substrate and substrate-analogues. FEBS Letters 307: 34-39 (1992)

Sup18. Perona, J.J., Hedstrom, L., Rutter, W.J., Fletterick,R.J. Structural Origins of Substrate Determination in Trypsin and Chymotrypsin. Biochemistry 34: 1489-1499 (1995)

Sup19. Helland, R., Leiros, I., Berglund, G.I., Willassen, N.P., Smalas, A.O. The Crystal Structure of Anioninc Salmon Trypsin in Complex with Bovine Pancreatic Trypsin Inhibitor European Journal of Biochemistry 256: 317-324 (1998)

Sup20. Wang, E.C., Hung, S.H., Cahoon, M., Hedstrom, L. The role of the Cys191-Cys220 disulfide bond in trypsin: new targets for engineering substrate specificity. Protein Engineering 10: 405-411 (1997)

Sup21. Fusarium oxysporum Trypsin at Atomic Resolution at 100 and 283K: A Study of Ligand Binding Acta Crystallographica Section D 57: 8-19 (2001)

Sup22. http://www.cryst.bbk.ac.uk/PPS2/course/section12/serprot3.html

Sup23. Sprang, S., Standing, T., Fletterick, R.J., Stroud, R.M., Finer-Moore, J., Xuong, N.H., Hamlin, R., Rutter, W.J., Craik, C.S. The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis. Science 237: 905-9 (1987)

Sup24. Ke, H., Zhang, Y., and Lipscomb, W.N. Crystal Structure of Fructose-1,6-bisphosphatase Complexed with Fructose 6-phosphate, AMP, and Magnesium. Proc. Natl. Acad. Sci. USA. 87: 5243-5247 (1990)

Sup25. Liang, J., Huang, S., Zhang, Y., Ke, H., Lipscomb, W.N. Crystal Structure of the Neutral Form of Fructose-1,6-bisphosphatase Complexed with Regulatory Inhibitor Fructose-2,6-bisphosphate at 2.6-A Resolution. Proc. Natl. Acad. Sci. USA. 89: 2404-2408 (1992)

Sup26. Xue, Y., Huang, S., Liang, J., Zhang, Y., and Lipscomb, W.N. Crystal Structure of Fructose-1,6-bisphosphatase Complexed with Fructose 2,6-bisphosphate, AMP, and Zn+2 at 2.0-A Resolution: Aspects of Synergism between Inhibitors. Proc. Natl. Acad. Sci. USA. 91: 12482-12486 (1994)

Sup27. Choe, J., Fromm, H.J., Honzatko, R.B. Crystal Structures of Fructose-1,6-bisphosphatase: Mechanism of Catalysis and Allosteric Inhibition Revealed in Product Complexes. Biochemistry 39: 8565-8574 (2000)

Sup28. Villeret, V., Huang, S., Fromm, H.J., Lipscomb, W.N. Crystallographic Evidence for the Action of Potassium, Thallium, and Lithium Ions on Fructose-1,6-bisphosphatase. Proc. Natl. Acad. Sci. USA. 92: 8916-8920 (1995)

Sup29. Schanstra, J.P., Ridder, I.S, Heimeriks, G.J., Rink, R., Poelarends, G.J., Kalk, K.H,, Dijkstra, B.W,, Janssen, D.B. Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range. Biochemistry 35(40):13186-95 (1996).

Sup30. Pries F, Kingma J, Krooshof GH, Jeronimus-Stratingh CM, Bruins AP, Janssen DB. 1995. Histidine 289 is essential for hydrolysis of the alkyl-enzyme intermediate of haloalkane dehalogenase. J Biol Chem 270:10405-10411 (1995).

Sup31. Pikkemaat, M. G., , Ridder, I.S., Rozeboom, H.J., Kalk,K.H., Dijkstra, B.W., and Janssen, D.B. Crystallographic and Kinetic Evidence of a Collision Complex Formed during Halide Import in Haloalkane Dehalogenase, Biochemistry 38, 12052-12072 (1999).

Sup32. Lipscomb, W.N., Strater, N. Recent advances in zinc enzymology. Chem Rev. 96, 2375-2433 (1996).

Sup33. Marie-Claire, C., Ruffet, E., Antonczak, S., Beaumont, A., O'Donohue, M., Roques, B. P., Fournie-Zaluski, M. C. Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding. Biochemistry. 36, 13938-13945 (1997).