title: Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.
abstract: Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of [14C]ATP or [14C]ADP in a manner that depends linearly on the extent of inactivation by either N,N'-p-phenylenedimaleimide or Co(II)phenanthroline/[Co(III)(phenanthroline)2CO3]+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 approximately 5 days). The nucleotide incorporated is not covalently bound because HClO4 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the gamma-phosphate of [gamma-32P]ATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-[beta, gamma-imido]triphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with N,N'-p-phenylenedimaleimide or cobalt phenanthroline complexes.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides; D009132:Muscles / Q000201:enzymology; D009218:Myosins / Q000378:metabolism*; D010618:Phenanthrolines; D011485:Protein Binding; D011817:Rabbits
chemical_list: D003432:Cross-Linking Reagents; D008301:Maleimides; D010618:Phenanthrolines; D000244:Adenosine Diphosphate; D000641:Ammonia; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D009218:Myosins