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

Abstract

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.

Wild Type and Mutant Human Heart (R)-3-Hydroxybutyrate Dehydrogenase Expressed in Insect Cells

“…BDH activity, expressed in Sf 9 cells, requires the addition of both NAD + and R-HOB for assay. Comparable activity was found using either only the R-isomer of HOB or the racemic mixture (RS-HOB) (not shown), as found previously for BH-BDH (Dalton et al, 1993). The addition of antimycin (used in mitochondria to block electron transport and re-oxidation of NADH) did not affect the measured BDH activity in lysed Sf 9 cells, consistent with lack of a specific mitochondrial localization of HH-BDH but rather a generalized distribution throughout the membrane fraction of the cells (see below).…”

“…The BDH activity of bovine heart submitochondrial vesicles (BH-SMV) was measured after addition of antimycin to inhibit reoxidation of NADH by electron transport. concentration (Dalton et al, 1993). At the highest multiplicity of infection, the specific activity of the expressed enzyme is comparable to that of BDH in bovine heart SMV (Table 1).…”

“…BDH activity in bovine heart submitochondrial vesicles was measured in the presence of 0.5 µg of antimycin A/mL to prevent oxidation of NADH by the electron-transport system. The apparent V max and K m values for R-HOB and NAD + were measured essentially as described (Dalton et al, 1993) using substrate and NAD + concentrations as indicated and with a total Na + of 220 mM. For CTT-D12, the R-HOB concentration was varied up to 500 mM with 20 mM NAD + .…”

Redox Modulation ofhsloCa2+-Activated K+Channels

“…This diversity of redox effects may be attributable to the reactivity of the sulfhydryl groups of cysteine residues; they may be alkylated, acylated, arylated, and oxidized to form some combination of sulfenic, sulfinic, or sulfonic compounds with varying stability. These adducts may contribute in complex ways to the covalent modification of redox modulatory sites (Dalton et al, 1993).…”

Characterization of Human DHRS6, an Orphan Short Chain Dehydrogenase/Reductase Enzyme

“…Mitochondrial R-␤-hydroxybutyrate dehydrogenase (BDH) 2 has been extensively studied (1)(2)(3)(4)(5) and constitutes a paradigm of lipid regulation of enzymatic function; however, a crystallographic structural characterization has not been achieved to date. The enzyme belongs to the short chain dehydrogenase/reductase superfamily (6 -8), an evolutionarily conserved family of oxidoreductases found in all forms of life.…”