The sulfhydryls of firefly luciferase are not essential for activity.
Firefly luciferase, containing an average of seven free sulfhydryls per two 50 000-dalton polypeptides, was modified by various sulfhydryl reagents. The differential reactivities of the sulfhydryls in luciferase protected by substrates allow one to define three categories of these groups: Class SH-III contains three sulfhydryls that are not involved in enzymatic activity. Class SH-II contains two sulfhydryls whose modification by different reagents causes varying effects on activity ranging from 0 to 60% inactivation. These sulfhydryls are not essential but may be important structurally or sterically. Class SH-I contains two sulfhydryls that are protected by substrates, either dehydroluciferyl adenylate or dehydroluciferin alone, and are located at or near the active site. The SH-I sulfhydryls are vicinal in the enzyme as demonstrated by their ability to form a disulfide bond. They have also been shown to exist on a single polypeptide chain. Modification of the SH-I groups by most reagents results in complete loss of enzymatic activity; reaction with methyl methanethiosulfonate produces an enzyme that emits only red light whereas native luciferase emits yellow-green light. Evidence is presented that the modified enzyme, while catalytically active, has a distorted active site. It is concluded that these two SH-I sulfhydryls are not essential for activity.
S C
SC
Alter
M
M
DeLuca
D001517:Coleoptera / Q000201:enzymology*; D005033:Ethylmaleimide / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008156:Luciferases; D005033:Ethylmaleimide