Role for an essential tyrosine in peptide amidation.
The catalytic core of the peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) domain of peptidylglycine alpha-amidating monooxygenase was investigated with respect to its ability to function as a ureidoglycolate lyase and the identity and role of its bound metal ions. The purified PAL catalytic core (PALcc) contains molar equivalents of calcium and zinc along with substoichiometric amounts of iron and functions as a ureidoglycolate lyase. Limiting iron availability in the cells synthesizing PALcc reduces the specific activity of the enzyme produced. Concentrated samples of native PALcc have an absorption maximum at 560 nm, suggestive of a phenolate-Fe(III) charge transfer complex. An essential role for a Tyr residue was confirmed by elimination of PAL activity following site-directed mutagenesis. Purified PALcc in which the only conserved Tyr residue (Tyr(654)) was mutated to Phe was secreted normally, but was catalytically inactive and lacked bound iron and bound zinc. Our data demonstrate an essential role for Tyr(654) and suggest that it serves as an Fe(III) ligand in an essential iron-zinc bimetallic site.
De
Mithu
M
Neuroscience Department, University of Connecticut Health Center, Farmington, Connecticut 06030-3401.
Bell
Joseph
J
Neuroscience Department, University of Connecticut Health Center, Farmington, Connecticut 06030-3401.
Blackburn
Ninian J
NJ
Neuroscience Department, University of Connecticut Health Center, Farmington, Connecticut 06030-3401.
Mains
Richard E
RE
Neuroscience Department, University of Connecticut Health Center, Farmington, Connecticut 06030-3401.
Eipper
Betty A
BA
Neuroscience Department, University of Connecticut Health Center, Farmington, Connecticut 06030-3401. Electronic address: eipper@uchc.edu.
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain; D006224:Cricetinae; D007501:Iron / Q000737:chemistry / Q000494:pharmacology; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry
D008024:Ligands; D009097:Multienzyme Complexes; D010455:Peptides; D014443:Tyrosine; D007501:Iron; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D015032:Zinc; D010100:Oxygen; D002118:Calcium