Conserved residues are functionally distinct within transketolases of different species.
Most of the amino acid residues which interact with thiamine pyrophosphate are highly conserved among enzymes which use this cofactor. The possible roles of several such residues in cofactor binding, catalysis, and/or substrate binding were examined for human transketolase. Mutations in H110 resulted in dramatic reductions to 2% or less of the normal activity. No alterations were found in the K(m)app's for the cofactor or for the donor and acceptor substrates. Alterations in Q428 resulted in a less severe loss of activity and also no changes in the K(m)app's. On the basis of the results, H110, an invariant residue, is proposed to function as a base which abstracts a proton from the protonated 4'-iminopyrimidine ring. The deprotonated 4'-imino moiety is required for generation of the C2-thiazolium carbanion which attacks the donor substrate. Interestingly, the function in the human enzyme of this invariant histidine is distinct from its role in yeast transketolase in which it aids in binding donor substrate and in subsequent catalytic events. Q428 is suggested to play a supportive role by stabilizing and orientating a water molecule which mediates the interaction between the 4'-amino group and H110. In other TPP-utilizing enzymes, the equivalent residue of Q428 is a histidine and is thought to deprotonate the 4'-amino group.
Singleton
C K
CK
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA. Singleck@ctrvax.Vanderbilt.edu
Wang
J J
JJ
Shan
L
L
Martin
P R
PR
D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014174:Transketolase / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014174:Transketolase; D013835:Thiamine Pyrophosphate