Conformational change of dihydrofolate reductase near the active site after thiol modification: detected by limited proteolysis.
Kinetic studies of chicken liver dihydrofolate reductase (CL-DHFR) and Chinese hamster ovary DHFR (CH-DHFR) activated following p-hydroxymercuribenzoate (p-HMB) modification indicate a conformational change at the active site, suggesting a loosening of the enzyme structure upon SH modification. In the present study, limited proteolysis was applied to detect the subtle conformational changes in SH-modified DHFRs. The digested peptide fragments were separated by Tricine SDS-PAGE and sequenced by Edman auto-degradation. The thiol modifier N-iodoacetyl-N'-(5-sulfo-1-nophthyl) ethylenediamine (IAEANS), which activates these DHFRs only weakly, was used as a control. The results of sequencing showed that compared to native enzyme, there is one additional cleavage site near the active site in p-HMB-modified CL-DHFR, two additional sites in p-HMB-modified CH-DHFR, but no additional site for IAEANS-modified DHFRs. These results indicate that activation of DHFRs following thiol modification is accompanied by a conformational change at or near the active site. This subtle change in the active site conformation results in a pronounced change in enzyme activity. This provides further evidence that flexibility at the active site is essential for full expression of enzyme catalytic activity. Comparing results obtained from previous experiments on guanidine- and urea-activated CL-DHFR, this shows that a conformational change near helix(28-39) is sufficient for full activation of DHFR.
Li
Z Y
ZY
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Science, 15 Datun Road, Chaoyang District, Beijing 100101, PR China.
Zhou
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D019791:Guanidine / Q000494:pharmacology; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000494:pharmacology; D010053:Ovary / Q000201:enzymology; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin
D006902:Hydroxymercuribenzoates; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C030601:4-hydroxymercuribenzoate; C006421:1,5-I-AEDANS; D013762:Tetrahydrofolate Dehydrogenase; D014357:Trypsin; D019791:Guanidine