On the molecular specificity of steroid-enzyme combinations. The kinetics ofβ-hydroxysteroid dehydrogenase

$\beta $-Hydroxysteroid dehydrogenase is a purified enzymic protein of bacterial origin which catalyzes oxidations of $3\beta -$ and $17\beta $-hydroxysteroids to their respective ketones with diphosphopyridine nucleotide as a hydrogen acceptor. The reaction kinetics of this enzyme with a variety of steroids are not in accordance with the predictions of the theory of Michaelis & Menten (1913), since the velocity of oxidation shows a marked tendency to decline at high concentrations of substrate. The behaviour of these compounds may be fully analyzed on the assumption of the formation of an enzyme-substrate complex involving two substrate molecules. The theory for bimolecular complex formation and its implications are examined. Affinity constants have been calculated for various steroids and conclusions drawn as to the structural requirements favouring attachment to the enzyme surface. Phenolic compounds of the oestra-1:3:5(10)-triene-3-ol family are most firmly bound. Planar molecules of the androst-4-ene, androst-5-ene or $5\alpha $-androstane series show intermediate affinity, while testane $(5\beta $-androstane) derivatives which deviate considerably from planarity are most poorly bound to the enzyme surface. The presence of oxygenated functions at positions 3 and 17 promotes high affinity, whereas an additional $11\alpha -$ or $11\beta $-hydroxyl group opposes this effect. Conclusions have been drawn as to the manner of attachment of substrates to the enzyme surface. Certain correlations between the molecular requirements for efficient binding of steroids to the enzyme surface and their physiological activities are demonstrated.