Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. Glutamic acid 418 in transketolase is essential for catalysis.
A homologous expression system and a purification protocol for pure, highly active recombinant yeast transketolase have been developed. The invariant transketolase residue Glu418, which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiamin diphosphate has been replaced by glutamine and alanine. Crystallographic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation. In both cases, the cofactor binds in a manner identical to the wild-type enzyme. Significant differences in the CD spectra of the mutant transketolases compared with the spectrum of wild-type enzyme indicate differences in the electron distribution of the aminopyrimidine ring of the cofactor. The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme. The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type transketolase. The hydrogen bond between the coenzyme and the side chain of Glu418 is thus not required for coenzyme binding but essential for catalytic activity. The results demonstrate the functional importance of this interaction and support the molecular model for cofactor deprotonation, the first step in enzymatic thiamin catalysis.
Wikner
C
C
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala.
Meshalkina
L
L
Nilsson
U
U
Nikkola
M
M
Lindqvist
Y
Y
Sundström
M
M
Schneider
G
G
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014174:Transketolase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D018698:Glutamic Acid; D014174:Transketolase; D013835:Thiamine Pyrophosphate