The demonstration of a glutamate dehydrogenase-NADP-L-glutamate charge-transfer complex and its location on the reaction pathway.

• DOI: 10.1016/s0021-9258(18)43921-x
• Publication Date: 2021-01-05T08:37:01Z
• Authors: S K Saha; S J Maniscalco; N Singh; H F Fisher
• Keywords: Glutamate synthase

In previous transient state kinetic work from this laboratory, we proposed a new mechanism for the glutamate dehydrogenase-catalyzed oxidative deamination reaction involving an initial replacement of proton lysine 126 by single bound water molecule, followed closure active site cleft and expulsion bulk water, providing hydrophobic environment ensuing hydride transfer step. Here, report results further fluorescence, absorbance, isotope effect studies, which demonstrate occurrence unusual intermediate in early steps that reaction. This phenomenon is revealed fluorescence burst occurs time period where absorbance signal still its lag phase. Using extension proton/product ratio approach have described earlier, show strongly fluorescent but weakly absorbing species whose absorption maximum red-shifted beyond other known complexes enzyme. The effects signals are compatible only with scheme formation complex precedes optical properties enzyme-oxidized coenzyme-substrate suggest it charge-transfer complex, similar nature to responsible well Racker band reported 1952 glyceraldehyde-3-phosphatase dehydrogenase (Racker, E., Krimsky, I. (1952) Nature 169, 1043-1044). crystal structure studies enzyme-coenzyme enzyme-L-glutamate closely analogous Clostridium symbosium dehydrogenase, Sheffield group (Stillman, T. J., Baker, P. Britton, K. L., Rice, D. W. (1993) J. Mol. Biol. 234, 1131-1139), provide basis physical explanation phenomenon. We conclude charge caused near apposition unprotonated alpha-amino substrate form enzyme conformational change has complete closing cleft.