CATALYTIC AND REGULATORY STRATEGIES OF THERMOPHILIC LACTATE-DEHYDROGENASE - MICROSCOPIC RATE CONSTANTS FROM KINETIC ISOTOPE EFFECTS

The lactate dehydrogenase of Bacillus stearrothermophilus exists in di meric and tetrameric forms, the latter favored by the regulatory effec tor, fructose-1,6-bisphosphate. The kinetic behavior of the two forms is different, the tetramer showing activation relative to the dimer at low pyruvate concentrations, but pyruvate inhibition at high concentr ations. Deuterium isotope effects at the transferring hydride site of NADH (primary isotope effect) and the methyl group of pyruvate (second ary isotope effects) allow estimation of the microscopic rate constant s for individual processes for both enzyme forms. In the dimer, entran ce and exit to and from the active site is slow for pyruvate nd lactat e, rapid for NAD. In the tetramer, entrance and exit for pyruvate and lactate are fast while NAD exit is slow. Hydride transfer occurs at si milar rates in both forms. This model explains the main features of th e kinetics of both forms of the enzyme.