Biochemistry 1990, 29, 10271-10280
10271
Combined Effects of Two Mutations of Catalytic Residues on the Ketosteroid Isomerase Reaction? Athan Kuliopulos,* Paul Talalay,* and Albert S. Mildvan*.* Department of Biological Chemistry and Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21 205 Received May 21, 1990; Revised Manuscript Received August 6, 1990
(EC 5.3.3.1) catalyzes the isomerization of A5-3-ketosteroids to A4-3-ketosteroids by a conservative tautomeric transfer of the 40-proton to the 60-position with Tyr- 14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1 989) Biochemistry 28, 149-1 591. Primary, secondary, and combined deuterium kinetic isotope effects establish concerted substrate enolization to be the rate-limiting step with the wild-type enzyme [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29,7491-75001. The product of the fractional k,, values resulting from the Y 14F mutation ( lo4.’) and the D38N mutation ( 1 0 - 9 is comparable ( 10-10.3) to that of the double mutant Y14F D38N (S10-’0.4) which is completely inactive. Hence, the combined effects are either additive or synergistic. Quantitatively, similar effects of the two mutations on kcat/KMare found in the double mutant. Despite its inactivity, the Y 14F D38N double mutant forms crystals indistinguishable in form from those of the wild-type enzyme, tightly binds steroid substrates and substrate analogues, and immobilizes a spin-labeled steroid in an orientation indistinguishable from that found in the wild-type enzyme, indicating that the double mutant is otherwise largely intact. It is concluded that the total enzymatic activity of ketosteroid isomerase probably results from the independent and concerted functioning of Tyr- 14 and Asp-38 in the rate-limiting enolization step, in accord with the perpendicular or antarafacial orientation of these two residues with respect to the enzyme-bound substrate. Synergistic effects of mutating two residues on k,, and on k,,/KM of enzyme-catalyzed multistep reactions are shown, theoretically, to occur when both residues act independently in the same step, and simple additivity occurs when this step is rate-limiting. Other conditions for additivity of the effects of mutations of k,,, and kat/& are theoretically explored.
ABSTRACT: A5-3-Ketosteroid isomerase
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T e enzyme As-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of As-3-ketosteroids to the conjugated A4-3-ketosteroids by a stereospecific and conservative transfer of the 40-proton to the 60-position by way of an enolic intermediate (Batzold et al., 1976; Bantia & Pollack, 1986) (Figure 1). Previous mechanistic studies (Kuliopulos et al., 1989) have established the importance of both Tyr-14 and Asp-38 in the isomerase reaction with the former serving as the proton donor (AH) and the latter as the proton acceptor (B) in the ratelimiting enolization step of the reaction (Xue et al., 1990). Model building studies based on X-ray and N M R data indicate these residues to be positioned in either a perpendicular or, less likely, antarafacial geometry with respect to the bound substrate, arrangements which are stereoelectronically appropriate for a concerted enolization of the substrate (Kuliopulos et al., 1989; Xue et al., 1990; Hand & Jencks, 1975; Rebek, 1988). Primary, secondary, and combined deuterium kinetic isotope effects have established the concerted enolization of the substrate as a major rate-limiting step in the overall reaction (Xue et al., 1990). Profound decreases in k,, by factors of lo4.’ and 105.6were reported for the Y 14F and D38N mutant enzymes, respectively. Indeed, the product of these factors (10’0.3)is comparable to the overall rate acceleration (-109.5) produced by the wild-type enzyme, as compared with the spontaneous rate of isomerization at the same temperature and pH, suggesting ‘This work was supported by NIH Grants DK 28616 (to A.S.M.) and DK 7422 (to P.T.). This work was begun by A.K. as a Trainee of the NIH Medical Scientist Training Program (T32 GM 7309). *Department of Biological Chemistry. Department of Pharmacology and Molecular Sciences.
0006-2960/90/0429-1027 1$02.50/0
that the entire catalytic power of this enzyme might be explained by the concerted action of these two residues in the hydrophobic site of the enzyme (Kuliopulos et al., 1989). As previously pointed out, however, before such quantitative conclusions can be drawn from the kinetic effects of mutations, it must be established that (1) the catalytic and structural effects of each mutation are limited to the residue altered and that (2) the contribution of each catalytic residue to the rate enhancement is independent from that of other catalytic residues and counted only once (Serpersu et al., 1987; Knowles, 1987; Carter et al., 1984; Fersht, 1987). The tight binding of substrates and substrate analogues by the Y14F and D38N single mutants (Kuliopulos et al., 1989) argues against major structural alterations beyond the residues mutated. However, these observations do not address whether the catalytic contributions of Tyr-14 and Asp-38 are independent and additive. The most direct way of testing this point is to determine how the effects of the individual mutations of these catalytic residues interact in a double mutant in which both catalytic residues have been altered (Ackers & Smith, 1985; Knowles, 1987; Carter et al., 1984; Weber et al., 1990). Accordingly, the double mutant Y 14F D38N was prepared, and its kinetic and substrate binding properties were studied. A preliminary report of this work has been published (Kuliopulos et al., 1990).
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EXPERIMENTAL PROCEDURES Materials. Spiro~doxyl-2,3’-5’a-androstanl17’B-01 (doxyl-DHT),’ mp 172-174OC, was obtained from Aldrich I Abbreviations: doxyl-DHT, spiro[doxyl-2,3’-5’cY-androstan]-l 7’8-01; BTP-CI or bis-Tris-propane-HCI, 1,3-bis[ [tris(hydroxymethyl)methyl]amino]propane dihydrochloride.
0 1990 American Chemical Society
10272 Biochemistry, Vol. 29, No. 44, 1990
Kuliopulos et al.
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Tyr 14-0-H u P 0& / H -%&i4 H' H
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