ENERGETICS IN ENZYMATIC CATALYSIS: TRIOSEPHOSPHATE ISOMERASE (DIFFUSION, MUTANT, ARCHAEBACTERIA, KINETICS)

(i) The essential catalytic base at the active site of the glycolytic enzyme triosephosphate isomerase (TIM) is the carboxylate group of Glu 165, which directly abstracts either the 1-pro-R proton of dihydroxyacetone 3-phosphate (DHAP) or the 2-proton of (R)-glyceraldehyde 3-phosphate (GAP) to yield the cis-enediol intermediate. The replacement of Glu 165 by Asp reduces the specific activity by a factor of about 1000. Comparison of the complete reaction energetics for the wild-type and mutant isomerases shows that only the free energies of the transition states for the two enolization steps have been seriously affected. (ii) TIM catalysis is diffusion controlled. With glycerol or sucrose as additive, the viscosity dependence of k(,cat)/K(,m) with DHAP or GAP as substrate is that expected for a diffusion-controlled process, with the Glu 165 to Asp mutant enzyme serving as control. Polymeric species increase the macroviscosity but not the microviscosity of the solution, and such additives do not affect the kinetics of the enzymatic reaction. (iii) TIM exists in two unliganded forms, one of which binds and isomerizes DHAP, and the other of which binds and isomerizes GAP. The tracer perturbation method of Britton demonstrates the kinetic significance of the interconversion of these two enzyme forms at high substrate concentrations, and yields a rate constant of approximately 106 s('-1) for this interconversion. (iv) TIM from Methanobacterium thermoautotrophicum, of the kingdom Archaebacteriae, has a specific catalytic activity of 10,000 units/mg and a k(,cat)/K(,m) of 3 x 10('8) M('-1)s('-1) with GAP as substrate. Comparison with the isomerases from eubacteria and from eukaryotes suggests that TIM may have evolved before the divergence of the three primary kingdoms. (v) The theoretical treatment of Albery & Knowles has been extended by describing the energetic consequences of maximizing in vivo flux for any enzyme operating in a metabolic or catabolic pathway. In a 'perfect' enzyme, the ratio of the concentrations of the Michaelis complexes is unity in the in vivo steady-state ( ep / es = 1). At equilibrium, this ratio is greater than unity ( ep (,o)/ es (,o) > 1), it increases with increasing in vivo irreversibility, and it is independent of the overall equilibrium constant.