Purification and Properties of NADP‐Dependent l(+)‐3‐Hydroxybutyryl‐CoA Dehydrogenase from Clostridium kluyveri

Abstract

l(+)‐3‐Hydroxybutyryl‐CoA dehydrogenase was purified 18 fold with a 29% yield from extracts of Clostridium kluyveri. The enzyme was found to be homogeneous by gel electrophoresis and ultracentrifugation. The analysis of the sedimentation equilibrium and gel filtration experiments gave a molecular weight of 220000 and 215000, respectively. Gel electrophoresis in the presence of sodium dodecylsulfate revealed the presence of 8 subunits per enzyme molecule. The subunits contain approx. 5 sulfhydryl groups per mole of protein (26000×g). l(+)‐3‐Hydroxybutyryl‐CoA dehydrogenase from C. kluyveri is NADP‐dependent. The enzyme does not oxidize 3‐hydroxyvaleryl‐CoA or 3‐hydroxycaproyl‐CoA. The specific activity of the pure enzyme as measured by acetoacetyl‐CoA reduction at the optimum pH of 6.5 is approx. 450 U/mg protein at 25°C. The oxidation of 3‐hydroxybutyryl‐CoA at pH 9.5 proceeds with 7% of the rate of acetoacetyl‐CoA reduction. N‐Acetyl‐S‐acetoacetyl‐cysteamine is reduced with 15% of the rate of acetoacetyl‐CoA. Measurements of the Michaelis constants for the substrates of l(+)‐3‐hydroxybutyryl‐CoA dehydrogenase gave values of 50 μM for acetoacetyl‐CoA and 70 μM. for NADPH2. Copyright © 1973, Wiley Blackwell. All rights reserved