MECHANISM OF THE REACTION CATALYZED BY DELTA(5)-3-KETOSTEROID ISOMERASE OF COMAMONAS (PSEUDOMONAS) TESTOSTERONI - KINETIC-PROPERTIES OF A MODIFIED ENZYME IN WHICH TYROSINE-14 IS REPLACED BY 3-FLUOROTYROSINE

Tyrosine 14 of Delta(5)-3-ketosteroid isomerase plays an important rol e in the function of the enzyme, since its replacement by phenylalanin e results in a decrease in k(cat) by a factor of 10(-4.7). This result and the fact that this residue resides in the enzyme's substrate bind ing site and is in close proximity to C-2 of the bound steroid suggest s that it functions as an electrophile in the catalytic mechanism by p rotonation of or hydrogen bonding to the C-3 carbonyl oxygen of the su bstrate. In order to obtain more information about the role of tyrosin e 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 pK(a)' of the pheno l hydroxyl should be decreased by about 1.5 log units. Site specificit y of this modification has been ensured by mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine. We find that re placement of tyrosine 14 by 3-fluorotyrosine in the Y55,88F modified f orm of the isomerase results in a 4-fold decrease in k(cat). We interp ret 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 inte rmediate in the overall reaction is dienol-like. An alternative mechan ism in which the intermediate is stabilized by a short, strong hydroge n bond can also be consistent with the data.