To build an enzyme.

Abstract

The structural components that lead to enzyme function are discussed for one simple enzyme-catalysed reaction: that mediated by triosephosphate isomerase. First, the recognition and binding of the substrates' phospho group is seen to involve four main-chain -NH- hydrogen bonds, two of which are positioned at the positive end of a short $\alpha $-helix aimed precisely at the phospho group and interact with the three peripheral phospho group oxygens. Second, the chemical steps (of substrate enolization) are shown to require both base and general acid catalysis. The identity and the positioning of the base, a carboxylate group, nicely fulfils the expectations both of mechanistic economy and of stereoelectronics. The identity of the general acid is shown by Fourier transform infrared and by $^{15}$N nuclear magnetic resonance (NMR) to be a neutral imidazole group, lying between the two substrate oxygens. The positioning of the ring is ideal, but its protonation state is unexpected. Thus the pK$_{\text{a}}$ of this histidine side-chain is < 4.5, lowered from 6.5 (the value in the denatured protein) by its position at the positive end of another well-aimed $\alpha $-helix. Third, the need for enzymes to provide kinetic barriers to the loss of reaction intermediates from the active site is emphasized. Triosephosphate isomerase achieves this sequestration of the reaction intermediate by using a flexible loop of the protein, and thus improves the efficiency of the catalysed transformation.