title: The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase: a kinetic study of the H172A mutant.
abstract: An essential histidine ligand to the electron transfer copper (CuH) of peptidylglycine alpha-hydroxylating monooxygenase (PHMcc) was mutated to an alanine and found to retain copper binding and hydroxylase activity [Jaron, S., et al. (2002) Biochemistry 41, 13274-13282]. An extensive kinetic and deuterium isotope effect study finds this mutant to maintain full coupling of O2 consumed to product formed despite a 3 order-of-magnitude decrease in kcat and a 300-fold decrease in kcat/Km(O2). Unexpectedly, electron transfer is not rate-limiting in H172A. Rather, the increased kinetic isotope effect (KIE) on kcat of 3.27 +/- 0.39 suggests that C-H bond cleavage has become more rate-limiting, implicating a role for His172 that goes beyond that of a simple ligand to CuH. The mechanistic implications are discussed.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720-1460, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Evans
authors.forename: Ninian J
authors.initials: NJ
authors.lastname: Blackburn
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D000409:Alanine / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D008024:Ligands; D009097:Multienzyme Complexes; D006639:Histidine; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000409:Alanine; D010100:Oxygen