A slow obligatory proton release step precedes hydride transfer in the liver glutamate dehydrogenase catalytic mechanism.
We have used the stopped-flow indicator dye method to measure proton release and product formation simultaneously in the initial transient-state portion of the glutamate dehydrogenase-catalyzed oxidative deamination of L-glutamate. We observe a measurably slow release of a proton from the enzyme-NADP-L-glutamate complex. This proton release precedes the hydride transfer step, as indicated by the distinct lag in the product formation signal. We show that the proton release step corresponds to an obligatory intermediate in the reaction sequence. We also find that compounds which are competitive inhibitors of L-glutamate are capable of inducing this phenomenon. We prove that this unanticipated prehydride transfer event cannot be due to the release of an alpha-amino group proton from the substrate.
Fisher
H F
HF
Department of Biochemistry, University of Kansas Medical Center, Kansas City, MO.
Maniscalco
S
S
Singh
N
N
Mehrotra
R N
RN
Srinivasan
R
R
D000818:Animals; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008962:Models, Theoretical; D009249:NADP / Q000378:metabolism; D011522:Protons; D013997:Time Factors
D011522:Protons; D009249:NADP; D005969:Glutamate Dehydrogenase