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

Pyridoxal 5'-phosphate (PLP) inactivated betaine aldehyde dehydrogenase (BA DH) from amaranth leaves in a time-dependent reversible process with biphas ic reaction kinetics. The initial rapid phase, which we could not resolve i n time, was followed by a slower phase with a second-order forward rate con stant 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 accumu lates during inactivation. The pH dependence of this second, slower phase o f the modification shows a pK of 8.0 +/- 0.1. Correlation studies between t he remaining activity and mol of PLP residues incorporated per mol of enzym e subunit after reduction with NaBH3CN suggested that there are two critica l lysyl groups for enzyme monomer. Both nucleotides, NAD(+) and NADH, provi ded significant protection against the slow phase of inactivation, but only NADH protected the enzyme against the first East phase. The PLP-modified e nzyme was not able to bind to an AMP-Sepharose column, suggesting that at l east one of the PLP-modified lysyl residues is involved in the binding of t he nucleotides. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.