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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Abstract
Studies of the proton-transfer reaction by P. testosteroni 3-oxo steroid .DELTA.4-.DELTA.5-isomerase with .DELTA.5(6)- and .DELTA.5(10)-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-.DELTA.5(6)-substrates have markedly high kcat. [catalytic rate constant] values, whereas those of 3-oxo-.DELTA.5(10)-substrates are very low and their apparent Km values approach equilibrium Kd. The 1st step in the isomerization process is: .**GRAPHIC**. [where E = enzyme, S = substrate] which is governed by the k-1/k+1 ratio and is very similar for the 2 classes of substrates (3-oxo-.DELTA.5(6)- and -.DELTA.5(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 the calculation of individual rate constants k+1 and k-1 and determination of 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. 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.