Phosphatidylcholine activation of human heart (R)-3-Hydroxybutyrate dehydrogenase mutants lacking active center sulfhydryls: Site-directed mutagenesis of a new recombinant fusion protein

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondria l enzyme with a specific requirement of phosphatidylcholine (PC) for functi on. A plasmid has been constructed to express human heart (HH) BDH in Esche richia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rap id two-step affinity purification yields active HH-Histag-BDH land six muta nts) with high specific activity (similar to 130 mu mol of NAD(+) reduced.m in(-1).mg(-1)). HH-Histag-BDH has no activity in the absence of phospholipi d and exhibits a specific requirement of PC for function. The HH-Histag-BDH -PC complex land HH-BDH derived therefrom by enterokinase cleavage) has app arent Michaelis constants (K-m values) for NAD+, NADH, (R)-3-hydroxybutyrat e (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat l iver BDH. A computed structural model of HH-BDH predicts the two active cen ter sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242, With both sulfydryls derivatized, BDH has minimal activity, but site-directed m utagenesis of C69 and/or C242 now shows that neither of these cysteines is required for PC activation or catalysis (the double mutant, C69A/C242A, is highly active with essentially normal kinetic parameters). Six cysteine mut ants each have an increased K-m(NADH) (2-6-fold) but an unchanged K-m(NAD+) . The C242S and C69A/C242S enzymes (but not the analogous C242A mutants nor the C69A or C69S mutants) exhibit similar to 10-fold increases in K-m(HOB) and K-m(AcAc), reflecting an altered substrate binding site. Thus, althoug h C242 tin the C-terminal lipid binding domain of BDH) is close to the acti ve site, it appears to be in a hydrophobic environment and only indirectly defines the substrate binding site at the catalytic center of BDH.