the –sh protection method for determining accurate kd values for enzyme-coenzyme complexes of nad+-dependent glutamate dehydrogenase and engineered mutants evidence for nonproductive nadph complexes英文论文.pdfVIP

SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 951472, 5 pages doi:10.4061/2010/951472 Research Article The –SH Protection Method for Determining Accurate Kd Values for Enzyme-Coenzyme Complexes of NAD+-Dependent Glutamate Dehydrogenase and Engineered Mutants: Evidence for Nonproductive NADPH Complexes Joanna Griffin and Paul C. Engel School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland Correspondence should be addressed to Paul C. Engel, paul.engel@ucd.ie Received 13 January 2010; Accepted 8 May 2010 Academic Editor: David Ballou Copyright © 2010 J. Griffin and P. C. Engel. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Inactivation rates have been measured for clostridial glutamate dehydrogenase and several engineered mutants at various DTNB concentrations. Analysis of rate constants allowed determination of Kd for each non-covalent enzyme-DTNB complex and the rate constant for reaction to form the inactive enzyme-thionitrobenzoate adduct. Both parameters are sensitive to the mutations + F238S, P262S, the double mutation F238S/P262S, and D263K, all in the coenzyme binding site. Study of the effects of NAD , NADH and NADPH at various concentrations in protecting against inactivation by 200 μM DTNB allowed determination of Kd values for binding of these coenzymes to each protein, yielding surprising results. The mutations were originally devised to lessen discrimina