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

Abstract

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 kcat. values, whereas those of 3-oxo-Δ5(10)-substrates are very low and their apparent Km 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 Km does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ‘catalytic perfection’.