The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase (PHM): a spectroscopic study of the H172A mutant.
The spectroscopic characterization of the H172A mutant of peptidylglycine alpha-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this Cu(H) ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 A in wild-type PHM to 1.89 A in H172A associated with a change in the structure of Cu(H) from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the Cu(M) site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at Cu(H) was obtained through the observation that loss of the H172 Cu(H) ligand caused a 3 cm(-)(1) blue shift in the nu(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.
Jaron
Shulamit
S
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921, USA.
Mains
Richard E
RE
Eipper
Betty A
BA
Blackburn
Ninian J
NJ
D000409:Alanine / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002248:Carbon Monoxide / Q000737:chemistry; D002384:Catalysis; D003300:Copper / Q000737:chemistry*; D006224:Cricetinae; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics*; D007093:Imidazoles / Q000737:chemistry; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics*; D009842:Oligopeptides / Q000737:chemistry; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013052:Spectrometry, X-Ray Emission; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis; D013379:Substrate Specificity / Q000235:genetics; D014965:X-Rays
D007093:Imidazoles; D008024:Ligands; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper; C029899:imidazole; D002248:Carbon Monoxide; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000409:Alanine