Search for: All records

The small magnitudes of some kinetic isotope effects (KIEs), including those associated with 13C, necessitate a highly precise experimental approach involving the competition of light and heavy substrates. Provided the reaction is first order in the labeled substrate, the product isotopologue ratio converges to the initial reactant isotopologue ratio at completion, but the same is not true for dimerization reactions simply because the product diverges into four distinct isotopologues. The relative populations of these dimers deviate from the statistical distribution under the influence of a KIE. Accordingly, the current study aims to demonstrate this concept by analyzing the relative 13C placement in D-alanine-D-alanine at reaction completion for the dimerization of D-[1-13C]alanine catalyzed by Mycobacterium tuberculosis D-alanine:D-alanine ligase (Ddl). Using 13C NMR spectroscopy and Fourier-transform ion cyclotron mass spectrometry, the relative distributions of the four dimer isotopologues were determined. The ratio of the mono-labeled dimers with 13C at the C-terminus to that with 13C at the N-terminus yielded a relative KIE of 1.011 ± 0.004 for the acyl carbon. This result suggests that the rate-limiting step of the Ddl-catalyzed reaction involves peptide bond formation—either nucleophilic attack by the amino group or collapse of the resulting tetrahedral intermediate. This method of analysis, to the best of our knowledge, is the first of its kind for obtaining competitive KIEs in enzyme-catalyzed dimerization reactions. 

Methylthio-D-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-D-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-D-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose–ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [32P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct. 

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) functions in the methionine salvage pathway by converting the cyclic aldose MTR1P to its open-chain ketose isomer methylthio-d-ribulose 1-phosphate (MTRu1P). What is particularly challenging for this enzyme is that the substrate’s phosphate ester prevents facile equilibration to an aldehyde, which in other aldose–ketose isomerases is known to activate the α-hydrogen for proton or hydride transfer between adjacent carbons. We speculated that MtnA could use covalent catalysis via a phosphorylated residue to permit isomerization by one of the canonical mechanisms, followed by phosphoryl transfer back to form the product. In apparent support of this mechanism, [32P]­MTR1P was found by SDS-PAGE and gel-filtration chromatography to radiolabel the enzyme. Susceptibility of this adduct to strongly acidic and basic pH and nucleophilic agents is consistent with an acyl phosphate. C160S and D240N, mutants of two conserved active-site residues, however, exhibited no difference in radiolabeling despite a reduction in activity of ∼107, leading to the conclusion that phosphorylation is unrelated to catalysis. Unexpectedly, prolonged incubations with C160S revealed up to 30% accumulation of radioactivity, which was identified by 31P and 13C NMR to be the result of a second adduct—a hemiketal formed between Ser160 and the carbonyl of MTRu1P. These results are interpreted as indirect support for a mechanism involving transfer of the proton from C-2 to C-1 by Cys160. 

Solvent isotope effects have long been used as a mechanistic tool for determining enzyme mechanisms. Most commonly, macroscopic rate constants such as kcat and kcat/Km are found to decrease when the reaction is performed in D2O for a variety of reasons including the transfer of protons. Under certain circumstances, these constants are found to increase, in what is termed an inverse solvent kinetic isotope effect (SKIE), which can be a diagnostic mechanistic feature. Generally, these phenomena can be attributed to an inverse solvent equilibrium isotope effect on a rapid equilibrium preceding the rate-limiting step(s). This review surveys inverse SKIEs in enzyme-catalyzed reactions by assessing their underlying origins in common mechanistic themes. Case studies for each category are presented, and the mechanistic implications are put into context. It is hoped that readers may find the illustrative examples valuable in planning and interpreting solvent isotope effect experiments.