A new procedure for deconvolution of inter-/intramolecular intrinsic primary and alpha-secondary deuterium isotope effects from enzyme steady-state kinetic data
Ws. Mcintire et al., A new procedure for deconvolution of inter-/intramolecular intrinsic primary and alpha-secondary deuterium isotope effects from enzyme steady-state kinetic data, J AM CHEM S, 121(25), 1999, pp. 5865-5880
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.