To build an enzyme

T he structural com ponents th a t lead to enzyme function are discussed for one simple enzyme-catalysed reac tio n : tha t m ediated by triosephosphate isomerase. First, the recognition and binding of the substrates’ phospho group is seen to involve four m ain-chain —N H — hydrogen bonds, two of which are positioned a t the positive end of a short a-helix aimed precisely at the phospho group and in teract with the three peripheral phospho group oxygens. Second, the chemical steps (of substrate enolization) are shown to require both base and general acid catalysis. T he identity and the positioning of the base, a carboxylate group, nicely fulfils the expectations both of m echanistic economy and of stereoelectronics. T he identity of the general acid is shown by Fourier transform infrared and by 15N nuclear m agnetic resonance ( n m r ) to be a neutral imidazole group, lying between the two substrate oxygens. T he positioning of the ring is ideal, but its p rotonation state is unexpected. Thus the pAa of this histidine side-chain is < 4.5, lowered from 6.5 (the value in the denatured protein) by its position a t the positive end of another well-aimed a-helix. T hird , the need for enzymes to provide kinetic barriers to the loss of reaction interm ediates from the active site is emphasized. Triosephosphate isomerase achieves this sequestration of the reaction interm ediate by using a flexible loop of the protein, and thus improves the efficiency of the catalysed transform ation.