Cyanylation of 3-Hydroxybutyrate Dehydrogenase. The “Essential” Sulfhydryl Group is not Involved in Catalysis

Abstract

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with 7V-ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [Mclntyre, J.O., Fleer, E.A.M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3 ; -dithiobis(6-nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited ~ 50% in excess substrate. Derivatization of the reactive sulfhy-dryl group results in loss of this substrate inhibition, the modified enzyme being at least threefold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the 5-cyanylated and SHcarboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of lOOmM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH-monomethylmalonate) (spin-labeled NAD®-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism.

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

“…In this regard, previous studies with BDH derivatized at SH-1 with 1 equiv. of cyanide or Ellman's reagent exhibited a similar enhancement in activity ( 3-fold) by high substrate [26] as found in the studies reported here for the DM derivative (BDH-II). Thus increased activity at high substrate is characteristic of SH-1modified BDH.…”

“…We find only small effects of SH-1 derivatization on coenzyme binding to BDH [2-fold or 5-fold increases in the apparent KmNAD+ by cyanylation [26] or diamide derivatization (Table 1)]. Thus there are limited effects of SH-1 derivatization on NAD+ binding to BDH, although it has been shown previously that: (1) bound NAD+ alters the reactivity of SH-1 [17,19,29]; and (2) the reactive thiol (SH-1) can be derivatized with arylazido-NAD+, indicating that it is near the active site [20].…”

“…Thus derivatization of SH-1 alters both the substrate-and cofactor-binding sites, with the reduction in enzymic activity being approximately proportional to the size of the derivatizing reagent. The effect of cyanylation of SH-1 of BDH on the kinetic parameters of the enzyme was to increase apparent KmBOH and Kmacetoacetate 10-fold and KmNAD+ and KmNADH about 2-fold [26]. We have now shown that derivatization of SH-1 with a larger reagent, diamide, has an even larger effect on the apparent KmBOH than does cyanylation (100-fold versus 10-fold increase), indicating that SH-1 is located at or near the substrate-binding site and consistent with the prediction [22] that the Cterminal domain provides specificity in substrate binding.…”

“…For DM derivatives of BDH, DTT was omitted from the enzyme assay. For comparison with previous studies [8,17,26], two fixed concentrations of Na+, 40 mM or 240 mM, were used with five variants (A-E) of the standard medium (see Figure 1). The enzymic reaction was initiated by addition of substrate and a computer program BDHACTV in HP-BASIC language was used for data acquisition and calculations (see Dalton [27]).…”

“…SH-1, whereas in the absence of NAD+ the stoichiometry of derivatization was one SH-1 per BDH protomer [19]; (4) alkylation of SH-1 perturbed energy transfer from an intrinsic tryptophan of BDH to bound NADH acceptor [19]; and (5) derivatization of the enzyme at SH-1 with reagents of variable size resulted in diminishing activity correlated with the size of the reagent [26]. Thus derivatization of SH-1 alters both the substrate-and cofactor-binding sites, with the reduction in enzymic activity being approximately proportional to the size of the derivatizing reagent.…”