Evaluation of the internal equilibrium constant for 3-oxo-delta 5-steroid isomerase using the D38E and D38N mutants: the energetic basis for catalysis.
The dissociation constant (KD) for the complex of the intermediate dienol (2) and the D38N mutant of 3-oxo-delta 5-steroid isomerase (D38N.2) has been determined for the isomerization of 5-androstene-3,17-dione (1). KD for D38N.2 is pH-dependent, with values of 6 nM at pH 6.9, 51 nM at pH 5.8, and 59 nM at pH 5.2. These values of KD are used to estimate the pH-independent dissociation constant (0.7 +/- 0.3 microM) for the complex of dienol and wild-type (WT) enzyme. The internal equilibrium constant (Kint = 0.3 +/- 0.2) for the interconversion of bound substrate (WT.1) and bound intermediate (WT.2) was then calculated for WT using its KD, the values for the external equilibrium constant for 1<-->2, and the dissociation constant of the enzyme substrate complex (KS). The dissociation constant (KD) for the complex of equilenin (4) with WT, D38E, and D38N enzymes was also determined at pH values from 4 to 7. For the complex of 4 with D38N (D38N.4), KD is pH-dependent with an apparent pKa of about 4.5, whereas KD for both WT.4 and D38E.4 is pH-independent. These values are used to give two additional estimates of the internal equilibrium constant for WT (Kint = 0.5 and 0.01). Analysis of these results in terms of Marcus formalism leads to the conclusion that the primary function of the enzyme is to decrease the thermodynamic barrier to formation of the intermediate by lowering delta Gzero by about 10 kcal/mol. In contrast, the intrinsic free energy of activation (delta G++int) is only decreased by about 3 kcal/mol. These results are discussed in terms of competing theories of enzymatic enolization.
Hawkinson
D C
DC
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228-5398.
Pollack
R M
RM
Ambulos
N P
NP
D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013251:Steroid Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D013251:Steroid Isomerases; C022660:steroid delta-isomerase