Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase.
The function of the mobile loop of triosephosphate isomerase has been investigated by deleting four contiguous residues from the part of this loop that interacts directly with the bound substrate. From the crystal structure of the wild-type enzyme, it appears that this excision will not significantly alter the conformation of the rest of the main chain of the protein. The specific catalytic activity of the purified mutant enzyme is nearly 10(5)-fold lower than that of the wild type. Kinetic measurements and isotopic partitioning studies show that the decrease in activity is due to much higher activation barriers for the enolization of enzyme-bound substrate. Although the substrates bind somewhat more weakly to the mutant enzyme than to the wild type, the intermediate analogue phosphoglycolohydroxamate binds much less well (by 200-fold) to the mutant. It seems that the deleted residues of the loop contribute critically to the stabilization of the enediol phosphate intermediate. Consistent with this view, the mutant enzyme can no longer prevent the loss of the enediol phosphate from the active site and its rapid decomposition to methylglyoxal and inorganic phosphate. Indeed, when glyceraldehyde 3-phosphate is the substrate, the enediol phosphate intermediate is lost (and decomposes) 5.5 times faster than it reprotonates to form the product dihydroxyacetone phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
Pompliano
D L
DL
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Peyman
A
A
Knowles
J R
JR
D002238:Carbohydrate Epimerases / Q000378:metabolism*; D003196:Computer Graphics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*
D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase