Cryoenzymological study of aspartate aminotransferase. Detection of intermediates by monitoring single turnovers with a true substrate.

The mechanism of action of mitochondrial aspartate aminotransferase has been investigated by cryoenzymological methods. For the first time a single half-reaction of enzymic transamination with a fast-reacting natural substrate could be monitored. The cryosolvent (50% methanol) did not affect the kinetic parameters for the overall reaction at 4 degrees C with cysteine sulfinate and oxaloacetate as substrates. The Km value for cysteine sulfinate at -44 degrees C, as determined from single-turnover experiments, was only slightly higher than that at 4 degrees C with and without cryosolvent. The kcat values obtained from analysis of the overall reaction at 4 degrees C to -33 degrees C give a linear Arrhenius plot (Ea = 87 kJ mol-1), which extrapolates to the kcat value estimated from single-turnover experiments at -44 degrees C. Apparently no change in the reaction path occurs over this large temperature range. On mixing pyridoxal enzyme and cysteine sulfinate at -44 degrees C, an intermediate absorbing at 430 nm was observed, which decayed in a biphasic process and most probably reflects the external aldimine. Under all conditions tested a build-up of a quninonoid intermediate was not observed, indicating that the protonation at C4' of the coenzyme is far from being rate-limiting and/or the equilibrium favors strongly the aldimine. The initial decay rate of the 430-nm intermediate indicates that this step might be partly rate-determining. However, the slower turnover rate as well as the shapes of intermediate spectra suggests another step, most likely the hydrolysis of the ketimine, to be actually rate-limiting.