Coenzyme binding of 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

Abstract

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)

Magnetic resonance of membranes

“…With this system, more functional studies are called for in order to clear up discrepancies whereas in other cases where the functional requirement for a particular lipid class is strong, e.g. of phosphatidylcholine for 3-hydroxybutyrate dehydrogenase (Rudy et al, 1989), more spin-labelling studies are needed. The links between specificity and function in proteinlipid interactions are discussed further by .…”

“…The alternative to spin-labelling the protein is to label a ligand, and studies on 3-hydroxybutyrate dehydrogenase using NAD(H) spin-labelled in the adenine ring offer a good illustration (Fritzsche et al, 1984;Rudy et al, 1989). 3-Hydroxybutyrate dehydrogenase is an enzyme that specifically requires phosphatidylcholine for activity.…”

Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase

“…The general methodology and analytical procedures were essentially as described previously [8,10,12,18], except as noted below. The thiol-group-derivatizing reagents, 1,1'-azobis-(NN'dimethylformamide) (diamide, DM), N-ethylmaleimide (NEM), and 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxyl (MSL) were obtained from Sigma Chemical Co. 7-Diethylamino-3-(4'maleimidophenyl)-4-methylcoumarin (CPM) and fluorescein-5-maleimide (FLM) were from Molecular Probes (Eugene, OR, U.S.A.).…”

“…Since the C-terminal domain of BDH provides substrate specificity and contains the arylazido-NAD+-reactive thiol (SH-1), the two domains would appear to be in close proximity in the folded (ternary or quaternary) structure [22]. In this regard the activation of BDH by PC is due in large part to PC-induced enhancement of nucleotide binding, i.e., a 50-fold decrease in the dissociation constant for NAD(H) in the presence versus the absence of PC [1 1,12]. Thus, although elements which constitute the PC-binding site are predicted to be in the C-terminal domain of BDH, activation of the enzyme by PC enhances nucleotide binding, the site for which is in the N-terminal domain of the protein.…”

Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme

“…(Isaacson et al, 1979), but bilayer association of BDH is not essential, since monomeric short-chain PC can also activate BDH (Gazzotti et al, 1975;Cortese et al, 1989). Activation of BDH by bilayer PC appears to involve an allosteric mechanism (Sandermann et al, 1986) in which PC enhances binding of nucleotide by more than an order of magnitude (Rudy et al 1989).…”