Previous studies of the mechanism of the steroid isomerase of Comamonas (Pseudomonas) testosteroni have identified aspartate 38 as the proton porter which transfers the substrate's 4 beta proton to the 6 beta position of the product. Consequently, aspartate 38 functions as a base in the deprotonation of the substrate to form a dienol or dienolate intermediate, which then undergoes reprotonation from protonated aspartate 38 at C-6 beta to give the product. We have tried to characterize the transition states for the proton transfers by altering the pKa' of aspartate 38 and then determining the effect of the alteration on the kinetics of the enzyme. Alteration of the pKa' was accomplished by replacement of the carboxyl carbon of aspartate 38 by sulfur, a change which converts the carboxylate group to the much less basic sulfinate group. Employing Brønsted catalysis theory as applied to the individual steps of the isomerase mechanism, we find that in the enolization step of the reaction proton transfer to aspartate 38 is well advanced in the transition state. In the subsequent ketonization step, proton transfer from aspartate 38 has barely started when that transition state is reached. A series of mutant KSIs with alternative bases at position 38 have been constructed using a combination of site-directed mutagenesis and chemical modification: Asp-38 to Glu (D38E), His (D38H), and S-(carboxymethyl)cysteine (D38CMC). While the D38H and D38E mutants both retain significant isomerase activity, D38CMC is essentially inert. From the results of kinetic experiments it is possible to get a qualitative idea of the sensitivity of the enzyme's catalytic ability to the location of the base responsible for proton transfer.

The structural gene coding for the A5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. pui biotype B genomic library in Xgtll. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986 A5-3-Ketosteroid isomerase (KSI) catalyzes the allylic isomerization of the 5,6 double bond of A5-3-ketosteroids to the 4,5 position by stereospecific intramolecular transfer of a proton (1) (Fig. 1) testosteroni KSI structure has been studied by X-ray crystallography. Three-dimensional structure of the KSI is known at 6-A (0.6-nm; 1 A = 0.1 nm) resolution (21). A preliminary, unrefined model has been obtained on the basis of a 2.5-A resolution electron density map (9). However, this model possesses an unacceptably high R-factor, and efforts to refine this structure in order to lower the R-factor have not been successful (hla). Part of the difficulty in solving the structure of C. testosteroni KSI arises from the very large size and anisotropy of the unit cell [P6 (1) The polypeptide of the KSI from P. putida biotype B is 6 residues longer than that of C. testosteroni KSI. As aligned, there are 44 identical matches, giving only 34% identity between two KSIs (11). A computer search of the sequence databases for proteins homologous to P. putida KSI has yielded no similar proteins other than C. testosteroni KSI. The critical active-site residues of C. testosteroni KSI, Asp-38 and Tyr-14, are conserved in the P. putida KSI polypeptide as Asp-40 and Tyr-16, respectively (reference 11 and this work).The P. putida KSI contains three cysteines and two tryptophans, whereas the C. testosteroni KSI lacks these amino acids.The P. putida KSI showed dependencies of Vm. and Km on pH that differ from those of C. testosteroni KSI (19). An activesite-directed photoinactivation study of the P. putida KSI implicated the modification of the sulfhydryl group in a cysteine, the extent of which correlated with the observed loss 6672 Vol. 21,No. 11

Versions of the Y55F/Y88F modified form of Delta 5-3-ketosteroid isomerase in which the active-site tyrosine-14 is replaced by 2-fluorotyrosine, 3-fluorotyrosine, and 2,3-difluorotyrosine, amino acids having progressively greater acidity of their phenolic hydroxyls, have been expressed in an Escherichia coli host and purified to high homogeneity. The steady-state kinetic properties of Y55F/Y88F KSI and its fluorotyrosine modified forms have been determined. The mechanistic implications of the results are presented and discussed.

Tyrosine 14 of delta 5-3-ketosteroid isomerase plays an important role in the function of the enzyme, since its replacement by phenylalanine results in a decrease in kcat by a factor of 10(-4.7). This result and the fact that this residue resides in the enzyme's substrate binding site and is in close proximity to C-2 of the bound steroid suggests that it functions as an electrophile in the catalytic mechanism by protonation of or hydrogen bonding to the C-3 carbonyl oxygen of the substrate. In order to obtain more information about the role of tyrosine 14, we have prepared a modified form of the enzyme in which tyrosine 14 has been substantially replaced in vivo by exogenously supplied 3-fluorotyrosine, a tyrosine derivative in which the pKa' of the phenol hydroxyl should be decreased by about 1.5 log units. Site specificity of this modification has been ensured by mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine. We find that replacement of tyrosine 14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in kcat. We interpret this result in terms of a mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from tyrosine 14 in the transition state, and the intermediate in the overall reaction is dienol-like. An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data.