Inactivation of betaine aldehyde dehydrogenase from amaranth leaves by pyridoxal 5′-phosphate

Abstract

Pyridoxal 5′-phosphate (PLP) inactivated betaine aldehyde dehydrogenase (BADH) from amaranth leaves in a time-dependent reversible process with biphasic reaction kinetics. The initial rapid phase, which we could not resolve in time, was followed by a slower phase with a second-order forward rate constant of 11.2 M −1 s −1 and a first-order reverse rate constant of 0.0012 s −1. The second, slower phase of inactivation was first order with respect to PLP and the rate increased linearly with PLP concentrations, suggesting that over the concentration range used no significant E-PLP complex accumulates during inactivation. The pH dependence of this second, slower phase of the modification shows a p K of 8.0±0.1. Correlation studies between the remaining activity and mol of PLP residues incorporated per mol of enzyme subunit after reduction with NaBH 3CN suggested that there are two critical lysyl groups for enzyme monomer. Both nucleotides, NAD + and NADH, provided significant protection against the slow phase of inactivation, but only NADH protected the enzyme against the first fast phase. The PLP-modified enzyme was not able to bind to an AMP-Sepharose column, suggesting that at least one of the PLP-modified lysyl residues is involved in the binding of the nucleotides.