Deuterium isotope effects substrate dependence

Use of deuterated substrates gives hAd = 6.5. This is a primary kinetic deuterium isotope effect, indicating diat proton removal is an essential component of die rate-determining step. The lack of rate dependence on bromine requires diat bromine is added to die molecule after die rate-determining step. A mechanism consistent widi diese facts has proton removal and enolate formation rate determining. 

Large primary kinetic isotope effects have been measured for the H-atom transfer steps from substrate to dAdo and from dAdo to the product radical in a number of AdoCbl-dependent enzymes as indicated in Table 19.1. In methylmalonyl-CoA mutase, the steady-state deuterium isotope effect is 5-6 in the forward direction, and the intrinsic isotope effect of step (i) in Scheme 19.3 is masked by the kineti-cally coupled but slower later steps [37-39]. The steady-state tritium kinetic isotope effect kii/kj) in the forward direction has been reported to be 3.2 [38]. Note that the experiments with deuterium were performed with a fully deuterated methyl group, while those with tritium were carried out at the trace level and correspond to a single isotopic atom therefore these two isotope effects should not be directly compared. For the reverse reaction, the deuterium kinetic isotope effect is also par-... 

Table 1. Substrate dependence of deuterium isotope effects ...

Zn > Co > Cd. Additionally, DHO activity is dependent on a single ionization for the dihydroorotate (favorable deprotonation) and carbamoyl aspartate (favorable protonation) substrates with apparent pA), values of 6 and 8.2, respectively. The pi), values change upon substitution with Co, suggesting that this reflects a metal-dependent ionization. There is a significant deuterium solvent isotope effect observed in /feat with the dihydroorotate ( cat / cat = 2.5 (ifcat/ M) /( cat/ M) = 1-1) and thio-dihydroorotate = 2.3 ... 

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