Cryoenzymological study of aspartate aminotransferase

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°C with cysteine sulfinate and oxaloacetate as substrates. The K m value for cysteine sulfinate at –44°C, as determined from single‐turnover experiments, was only slightly higher than that at 4°C with and without cryosolvent. The k cat values obtained from analysis of the overall reaction at 4°C to –33°C give a linear Arrhenius plot ( E a = 87 kJ mol −1 ), which extrapolates to the k cat value estimated from single‐turnover experiments at –44°C. Apparently no change in the reaction path occurs over this large temperature range. On mixing pyridoxal enzyme and cysteine sulfinate at –44°C, an intermidiate 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.