Enzyme substrate complex

Abstract: Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.

Abstract: A simple derivation is given that the catalytic term k$\_{\text{cat}}$/K$\_{\text{S}}\dagger $ is at a maximum when the structure of the enzyme is complementary to the structure of the substrate in the transition state. In addition, at a constant substrate concentration, [S], the maximum reaction rate is obtained when k$\_{\text{cat}}$ and K$\_{\text{S}}$ are individually high so that K$\_{\text{S}}$ is greater than [S]; the overall reaction rate decreases with decreasing k$\_{\text{cat}}$ and K$\_{\text{S}}$ for K$\_{\text{S}}$ less than [S]. Two corollaries of this are that intermediates accumulating after the initial Michaelis complex are undesirable and also enzymes whose function is to optimize reaction rates should have evolved to exhibit K$_{\text{M}}$ values above those of accessible substrate concentrations. This could be achieved by an often 'distortionless' strain which consists either of unfavourable interactions in the enzyme substrate complex which are relieved in the transition state or increasingly favourable interactions in the transition state. A possible special role of the backbone NH groups in this context is discussed. The enzyme need not be complementary to the transition state of the substrate for catalysis to occur.