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 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 k(cat.) values, whereas those of 3-oxo-Delta(5(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-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 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'.

Comparison of Two Unusual Enoyl-CoA Reductases in Streptomyces collinus

“…Further support for this came from the gc-ms analyses of the cell-free incubation products which revealed that under the conditions of the experiment greater than 50% of the racemic 6 reacted. This result is similar to the partial regiospecific promiscuity that has been observed (29,30) between two carbons in the reprotonation step of A5-3-ketosteroid isomerases (in this case the catalytic group has been shown to be a freely rotating carboxylate of an aspartate residue).…”

Mechanistic Studies of a Δ1, Δ2 Cyclohexenylcarbonyl CoA Isomerase Catalyzing the Penultimate Step in the Biosynthesis of the Cyclohexanecarboxylic Acid Moiety of Ansatrienin A

“…This too was confirmed by the gc-ms analyses of the cell-free incubation products, which revealed that under the conditions of the experiment greater than 50% of the racemic 3 reacted. This result is similar to the partial regiospecific promiscuity that has been observed between two carbons in the reprotonation step of A5-3-ketosteroid isomerases [in this case the catalytic group has been shown to be a freely rotation carboxylate of an aspartate residue (10,11)].…”

Kinetic and ultraviolet spectroscopic studies of active-site mutants of .DELTA.5-3-ketosteroid isomerase

“…The kinetic data were analyzed by double reciprocal plots, and the Km and FmaJt values were obtained by use of a Wilkinson hyperbolic weighted leastsquares program (Wilkinson, 1961). The differing volumes of methanol used in the above experiments were dictated by a compromise between the inhibition of the enzyme by this solvent (Weintraub et al, 1980;Falcoz-Kelly et al, 1968) and the need to maintain steroid solubility.…”