PMID- 10190977
OWN - NLM
STAT- MEDLINE
DCOM- 19990604
LR  - 20131121
IS  - 0003-9861 (Print)
IS  - 0003-9861 (Linking)
VI  - 364
IP  - 2
DP  - 1999 Apr 15
TI  - Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti 
      conformation of NAD+ nicotinamide but does not experience A-specific hydride 
      transfer.
PG  - 219-27
AB  - Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent 
      oxidoreductase which catalyzes the oxidative phosphorylation of 
      d-glyceraldehyde-3-phosphate (G3P) to form 1, 3-diphosphoglycerate. The currently 
      accepted mechanism involves an oxidoreduction step followed by a phosphorylation. 
      GAPDH is classified as a B-specific oxidoreductase. The inspection of several 
      crystal structures of GAPDHs indicates that the efficient hydride transfer from 
      the hemithioacetal intermediate to the C4 position of the pyridinium si face 
      requires optimal nicotinamidium-protein contacts for a suitable pyridinium-ring 
      orientation. In previous studies carried out on Escherichia coli GAPDH (C. 
      Corbier, A. Mougin, Y. Mely, H. W. Adolph, M. Zeppezauer, D. Gerard, A. Wonacott, 
      and G. Branlant, Biochimie 72, 545-554, 1990; J. Eyschen, C. Corbier, B. Vitoux, 
      G. Branlant, and M. T. Cung, Protein Pept. Lett. 1, 19-24, 1994), the role of the 
      invariant Asn 313 residue, as an anchor which favors the syn orientation of the 
      nicotinamide ring, was examined. Here, we report further investigations on the 
      molecular factors responsible for the cofactor stereospecificity. Two single 
      [Gly317] and [Ala317] GAPDH mutants and one double [Thr313-Gly317] GAPDH mutant 
      were constructed on the basis of a molecular modelling study from the crystal 
      structure of holo GAPDH from E. coli (E. Duee, L. Olivier-Deyris, E. Fanchon, C. 
      Corbier, G. Branlant, and O. Dideberg, J. Mol. Biol. 257, 814-838, 1996). The Kd 
      constants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAD are 5, 
      13, and 300 times higher than that of wild-type GAPDH. Transferred nuclear 
      Overhauser effect spectroscopy demonstrates that the wild-type syn orientation of 
      bound nicotinamide remains unchanged in the [Gly317] and [Ala317] mutants, 
      whereas a conformational equilibrium between the syn and anti forms occurs in the 
      [Thr313-Gly317] double mutant with a preference for the anti conformer. Although 
      the double mutant preferably binds the nicotinamide ring in an anti conformation, 
      it still exhibits B hydride transfer stereospecificity. Yet, the catalytic 
      efficiency is much less than that of the wild type. This indicates that the holo 
      GAPDH mutant fraction with an anti orientation of bound NAD is not capable of 
      forming the ternary complex with G3P which would be required for an efficient 
      A-specific catalytic process. The reasons of this catalytic inefficiency are 
      discussed in relation with the historical and functional models which were 
      advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases.
CI  - Copyright 1999 Academic Press.
FAU - Eyschen, J
AU  - Eyschen J
AD  - UMR 7567 CNRS-UHP, Maturation des ARN et Enzymologie Moleculaire, B. P. 239, 
      Vandoeuvre-les-Nancy, 54506, France.
FAU - Vitoux, B
AU  - Vitoux B
FAU - Marraud, M
AU  - Marraud M
FAU - Cung, M T
AU  - Cung MT
FAU - Branlant, G
AU  - Branlant G
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Arch Biochem Biophys
JT  - Archives of biochemistry and biophysics
JID - 0372430
RN  - 0U46U6E8UK (NAD)
RN  - EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
SB  - IM
MH  - Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry/genetics/*metabolism
MH  - Ion Transport
MH  - Magnetic Resonance Spectroscopy
MH  - Mutagenesis, Site-Directed
MH  - NAD/chemistry/*metabolism
MH  - Protein Conformation
MH  - Stereoisomerism
MH  - Substrate Specificity
EDAT- 1999/04/07 00:00
MHDA- 1999/04/07 00:01
CRDT- 1999/04/07 00:00
PHST- 1999/04/07 00:00 [pubmed]
PHST- 1999/04/07 00:01 [medline]
PHST- 1999/04/07 00:00 [entrez]
AID - S0003-9861(99)91116-5 [pii]
AID - 10.1006/abbi.1999.1116 [doi]
PST - ppublish
SO  - Arch Biochem Biophys. 1999 Apr 15;364(2):219-27. doi: 10.1006/abbi.1999.1116.