Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

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Abstract
Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD+ and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD+ and propionaldehyde with the enzyme were varied were fitted to the following mechanism: .**GRAPHIC**. where the release of NADH was slow. By monitoring the quenching of protein fluorescence on the binding of NAD+, estimates of 2 .times. 105 l .cntdot. mol-1 .cntdot. and 2s-1 were obtained for the rate constants k+1 and k-1 respectively. Although k+3 could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s-1, k+2 and k-2 could not be determined uniquely, but could be related by the equation: (k-2+k+3)/k+2 = 50 .times. 10-6 mol .cntdot. l-1. No significant isotope effect was observed when [1-2H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k-2 + k+3)/k+2 = 2.3 .times. 10-3 mol .cntdot. l -1 and k+3 is 23s-1. When [1,2,2,2-2H]acetaldehyde was used, no isotope effect was observed on k+3, but there was a significant effect on k+2 and k-2. A burst of NADH production was also observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.