Understanding protein lids: kinetic analysis of active hinge mutants in triosephosphate isomerase.
In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.
Sun
J
J
Department of Chemistry, State University of New York, Stony Brook 11794-3400, USA.
Sampson
N S
NS
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002645:Chickens; D003903:Deuterium / Q000737:chemistry; D004099:Dihydroxyacetone Phosphate / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D014305:Triose-Phosphate Isomerase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D004099:Dihydroxyacetone Phosphate; D003903:Deuterium; D014305:Triose-Phosphate Isomerase