A new procedure for deconvolution of inter-/intramolecular intrinsic primary and alpha-secondary deuterium isotope effects from enzyme steady-state kinetic data

The A(2)B(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudom onas putida oxidizes 4-methylphenol (p-cresol) to 4-hydroxybenzyl alcohol i n a process requiring scission of an a-C-H bond with concomitant reduction of covalently bound FAD in each A subunit. Values of k(cat)/K were determin ed from steady-state kinetic data for the reactions of PCMH with the follow ing substrates: 4-methylphenol, 4-(H-2(1))methylphenol, 4-(H-2(2))methylphe nol, and 4-(H-2(3))methylphenol. A procedure was devised to extract the int rinsic primary deuterium and intrinsic alpha-secondary deuterium kinetic is otope effects from these values of k(cat)/K. The primary effect, P, is 6.71 +/- 0.08, and the secondary effect, S, is 1.013 +/- 0.014. The magnitudes of these effects are discussed in terms of an early or late transition stat e, hydrogen tunneling, coupled motion between the leaving and remaining hyd rogens of the methyl group, and a H- expulsion mechanism versus a substrate radical mechanism versus a covalent substrate-FAD intermediate mechanism. The reaction of 4-ethylphenol with PCMH produces 4-vinylphenol and (-)-S-1- (4-hydroxyphenyl)ethanol (similar to 100% enantomeric excess). The evidence indicates that these are formed from a common intermediate, presumably a p -quinone methide. From the partition ratios for the formation of the alcoho l and 4-vinylphenol from 4-ethylphenol, 4-(1',1'-H-2(2))ethylphenol, and 4- (2',2',2'-H-2(3))ethylphenol, the primary isotope effect for conversion of the p-quinone (2',2',2' 2H3)methide to 4-(2',2'-H-2(2))vinylphenol was esti mated to be about 2, and the a-secondary isotope effect for conversion of p -quinone (1'-H-2(1))methide to 1-(4-hydroxyphenyl)-(1'-H-2(1))ethanol was f ound to be inverse (=0.83), as expected for sp(2) to sp(3) hybridization ch ange at the alpha-carbon. Values of k(cat)/K were determined for 4-ethylphe nol, R,S-(+/-)-4-(1'-H-2(1))ethylphenol (abbreviated R,S-D), S-(-)-4-(1'-H- 2(1))ethylphenol (S-D), R-(+)4-(1'-H-2(1))ethylphenol (R-D), and 4-(1',1'-H -2(2))ethylphenol (D2). The (D2)(k(cat)/K) value was found to be 5.1-6.1, t he same as determined in an earlier study. Unexpectedly, the values for (R, S-D)(k(cat)/K), (S-D)(k(cat)/K), and (R-D)(k(cat)/K) were all about the sam e (similar to 1.7), indicating that then is nearly an equal probability for pro-R or pro-S C-H bond scission. An apparent flux ratio for the pro-S pat h/pro-R path was estimated to be 0.78 +/- 0.02. The same procedure devised to determine values for P and S for 4-methylphenol was used to determine th ese values for the 4-ethylphenol reaction (commitment to catalysis = 0); P = 5.98 +/- 0.12 and S = 0.967 +/- 0.021. These values are essentially the s ame as those determined for 4-methylphenol. Thus, the chemical mechanisms f or both substrates are assumed to be similar.