CONSERVED RESIDUES ARE FUNCTIONALLY DISTINCT WITHIN TRANSKETOLASES OFDIFFERENT SPECIES

Most Of the amino acid residues which interact with thiamine pyrophosp hate are highly conserved among enzymes which use this cofactor. The p ossible roles of several such residues in cofactor binding, catalysis, and/or substrate binding were examined for human transketolase. Mutat ions in H110 resulted in dramatic reductions to 2% or less of the norm al activity. No alterations were found in the K(m)app's for the cofact or or for the donor and acceptor substrates. Alterations in Q428 resul ted in a less severe loss of activity and also no changes in the K(m)a pp's. On the basis of the results, H110, an invariant residue, is prop osed to function as a base which abstracts a proton from the protonate d 4'-iminopyrimidine ring. The deprotonated 4'-imino moiety is require d for generation of the C2-thiazolium carbanion which attacks the dono r substrate. Interestingly, the function in the human enzyme of this i nvariant 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 t he 4'-amino group and H110. In other TPP-utilizing enzymes, the equiva lent residue of Q428 is a histidine and is thought to deprotonate the 4'-amino group.