Influence of the position of the double bond in steroid substrates on the efficiency of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Δ⁴–Δ⁵-isomerase

Studies of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Δ⁴–Δ⁵-isomerase with Δ⁵⁽⁶⁾- and Δ⁵⁽¹⁰⁾-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-Δ⁵⁽⁶⁾-substrates have markedly high k_cat. values, whereas those of 3-oxo-Δ⁵⁽¹⁰⁾-substrates are very low and their apparent K_m values approach equilibrium dissociation constants. The first step in the isomerization process is: [Formula: see text] which is governed by the k₋₁/k₊₁ ratio and is shown to be very similar for the two classes of substrates (3-oxo-Δ⁵⁽⁶⁾- and -Δ⁵⁽¹⁰⁾-steroids). They therefore differ in the steps distal to the initial formation of the Michaelis–Menten complex. The use of the deuterated androst-5(6)-ene-3,17-dione substrate enabled us to calculate individual rate constants k₊₁ and k₋₁ as well as to determine the apparent rate-limiting step in the isomerization process. With the deuterated oestr-5(10)-ene-3,17-dione substrate, no significant isotope effect was observed suggesting that a different rate-limiting step may be operative in this isomerization process. Data are presented that indicate that under optimal concentrations of the efficient androst-5(6)-ene-3,17-dione substrate, the forward reaction for ES complex formation (as defined by k₊₁) is limited only by diffusion and the apparent K_m does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ‘catalytic perfection’.