internal competition method using isotope ratio mass spectrometry. Therefore the results
obtained represent the isotopic substitution effects on V/Koxaiate, and are associated with
the first irreversible step of the mechanism and all the steps leading to it.34, 35 Analyzed
CO2(g) was obtained from direct isolation from the oxalate decarboxylase, catalyzed
partial conversion of oxalate or from the oxidation of initial oxalate, format and oxalate
from residual substrate after the partial reaction in anhydrous DMSO with iodine.
Isotopic ratios obtained are the R values (defined under the equation below) for these
compounds. Analysis of KIE results for mutated OxDc was based on a literature equation
for the wild type enzyme described below.13,41
ln(1 f) n(1 f)
applE = =
ln (1-f) Rs] n l[1-fR
Ro Ro
13or1 (V / K)C02 ( + x)appIE
2x
13or18 (V / K) format = x) appIE
x = Rc0 / Rformate
Rp = (Rco2 + Rformate )/ 2
f= fraction of reaction
R isotopic ratio determined by MS for:
Ro = initial oxalate
Rs = residual substrate
Rco2 = produced CO2
Rformate = produced format