Bacterial
5-3-ketosteroid isomerase (KSI) catalyzesa stereospecific isomerization of steroidsubstrates at an extremely fast rate, overcoming a large disparity ofp
Ka values between a catalyticresidueand its target. The crystal structures of KSI from
Pseudomonas putida and of the enzyme in complexwith equilenin, an analogue of the reaction intermediate, have beendetermined at 1.9 and 2.5 Å resolution,respectively. The structures reveal that the side chains ofTyr
14 and Asp
99 (a newly identifiedcatalyticresidue) form hydrogen bonds directly with the oxyanion of the boundinhibitor in a completely apolarmilieu of the active site. No water molecule is found at theactive site, and the access of bulk solvent isblocked by a layer of apolar residues. Asp
99 issurrounded by six apolar residues, and consequently, itsp
Ka appears to be elevated as high as 9.5 to beconsistent with early studies. No interaction wasfoundbetween the bound inhibitor and the residue 101 (phenylalanine in
Pseudomonas testosteroni andmethioninein
P. putida KSI) which was suggested to contributesignificantly to the rate enhancement based onmutational analysis. This observation excludes the residue 101 asa potential catalytic residue and requiresthat the rate enhancement should be explained solely byTyr
14 and Asp
99. Kinetic analyses of Y14FandD99L mutant enzymes demonstrate that Tyr
14 contributes muchmore significantly to the rate enhancementthan Asp
99. Previous studies and the structuralanalysis strongly suggest that the low-barrier hydrogenbond of Tyr
14 (>7.1 kcal/mol), along with a moderatestrength hydrogen bond of Asp
99 (~4kcal/mol),accounts for the required energy of 11 kcal/mol for thetransition-state stabilization.