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’.
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
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Hadassa Weintraub, Etienne-Emile Baulieu, Annette Alfsen; 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. Biochem J 1 March 1980; 185 (3): 723–732. doi: https://doi.org/10.1042/bj1850723
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