Magnetic resonance studies of specificity in binding and catalysis of phosphotransferases.
Two common characteristics of the active site structures of intermediate complexes formed in kinase reactions have been observed by magnetic resonance techniques. First, in creatine, arginine, adenylate and pyruvate kinases (EC2.73.2, 2.7.3.3, 2.7.4.3 and 2.7.1.40, respectively) water is progressively excluded and the structure at the active site is progressively immobilized as each reactant is successively added to the enzyme, as monitored by electron spin resonance (e.s.r) and the enhancement of the proton relaxation rate of water (PRR) due to paramagnetic manganese(II) probe. Significant, and often wide-spread, changes in the protein conformation accompanying successive additions of reaction components are shown with 1H n.m.r. studies of pyruvate kinase. The second characteristic is that, for the ternary enzyme-Mn-nucleotide complexes, two parameters, the e.s.r. spectrum and PRR enhancement values, fall within a range of 10% for all enzymes investigated, with the exception of bovine brain creatine kinase. These similarities suggest a homology in teriary structure at the active sites of these enzymes. An unsuspected aspect of substrate and cofactor specificity has been revealed by e.s.r. spectroscopy of the manganese(II) complexes of the transition-state analogue of creatine kinase (E-MnADP-formate-creatine) and of the ternary phosphoenolpyruvate complex. In the former case, replacement of ADP, the normal substrate, by its substrate analogues IDP or 2acute-deoxyadenosine diphosphate produced two interconvertible species of the transition-state analogue complexes, observed in the e.s.r. spectra as an isotropic species and a highly anisotropic species. With the normal substrate, only the anisotropic species is observed. Similarly, in the case of the complex pyruvate kinase-Mn-phosphoenolpyruvate, when the normal monovalent activator K+ is replaced by the inert tetramethylammonium ion, again two interconvertible species rather than the normal one species are observed by e.s.r. spectroscopy. The implications of these phenomena for the relation of specificity to catalytic efficiency are discussed.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D002417:Cattle; D003402:Creatine Kinase / Q000378:metabolism; D004735:Energy Transfer; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D009132:Muscles / Q000201:enzymology; D010770:Phosphotransferases / Q000378:metabolism*; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000378:metabolism; D011817:Rabbits; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine; D013696:Temperature; D014867:Water / Q000378:metabolism
D013438:Sulfhydryl Compounds; D014867:Water; D008345:Manganese; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D010770:Phosphotransferases; D011770:Pyruvate Kinase; D003402:Creatine Kinase; D000263:Adenylate Kinase; D008274:Magnesium