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 mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity ( approximately 130 micromol of NAD(+) reduced.min(-1).mg(-1)). HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function. The HH-Histag-BDH-PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (K(m) values) for NAD(+), NADH, (R)-3-hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH. A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242. With both sulfhydryls derivatized, BDH has minimal activity, but site-directed mutagenesis 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 mutants 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 approximately 10-fold increases in K(m)(HOB) and K(m)(AcAc), reflecting an altered substrate binding site. Thus, although C242 (in the C-terminal lipid binding domain of BDH) is close to the active site, it appears to be in a hydrophobic environment and only indirectly defines the substrate binding site at the catalytic center of BDH.