Correction of pre-steady-state KIEs for isotopic impurities and the consequences of kinetic isotope fractionation.
We show, both experimentally and by kinetic modeling, that enzymatic single-turnover (pre-steady-state) H-transfer reactions can be significantly complicated by kinetic isotope fractionation. This fractionation results in the formation of more protiated than deuterated product and is a unique problem for pre-steady-state reactions. When observed rate constants are measured using rapid-mixing (e.g., stopped flow) methodologies, kinetic isotope fractionation can lead to a large underestimation of both the magnitude and temperature dependence of kinetic isotope effects (KIEs). This fractionation is related to the isotopic purity of the substrates used and highlights a major problem with experimental studies which measure KIEs with substrates that are not isotopically pure. As it is not always possible to prepare isotopically pure substrates, we describe two general methods for the correction, for known isotope impurities, of KIEs calculated from pre-steady-state measurements.
Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
Sam
S
Hay
Christopher R
CR
Pudney
Parvinder
P
Hothi
Nigel S
NS
Scrutton
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001596:Benzylamines / Q000378:metabolism; D002384:Catalysis; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D007554:Isotopes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D001596:Benzylamines; D003067:Coenzymes; D004798:Enzymes; D007554:Isotopes; D009249:NADP; C030796:benzylamine; D010088:Oxidoreductases; C087820:morphinone reductase; D000587:Oxidoreductases Acting on CH-NH Group Donors; C037193:aromatic amine dehydrogenase; C098939:pentaerythritol tetranitrate reductase