ALKYLATION AT THE ACTIVE-SITE OF THE D-3-HYDROXYBUTYRATE DEHYDROGENASE (BDH), A MEMBRANE PHOSPHOLIPID-DEPENDENT ENZYME, BY 3-CHLOROACETYL PYRIDINE ADENINE-DINUCLEOTIDE (3-CAPAD)

The structure of the rat liver's D-3-hydroxybutyrate dehydrogenase (BD H) active site has been investigated using an affinity alkylating reag ent, the 3-chloroacetyl pyridine adenine dinucleotide (3-CAPAD). This NAD(+) analogue reagent strongly inactivates the enzyme following a co ncentration- and time-dependent process with a stoichiometry of approx imately 1. The reagent reacts at the coenzyme binding site as revealed by the efficient protection by NADH. The effect of 3-CAPAD is stronge r with the enzyme into its natural membrane environment than with the lipid-free purified apoBDH or with the reconstituted apoBDH-mitochondr ial phospholipid complex. The pH-dependent effect on the inactivation process is in agreement with the participation of protons in the catal ytic mechanism of BDH. Furthermore, this study exhibits the phospholip id activating role in BDH catalytic activation.