The inactivation of bifunctional peptidylglycine alpha-amidating enzyme by benzylhydrazine: evidence that the two enzyme-bound copper atoms are nonequivalent.
Peptidylglycine alpha-amidating enzyme catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate in a reaction that requires O2, ascorbate and 2 mol of copper per mole of enzyme [Kulathila et al. (1994) Arch. Biochem. Biophys. 311, 191-195]. Peptides with a C-terminal alpha-hydroxyglycine residue are intermediates in the amidation reaction. Benzylhydrazine inactivates the enzymatic conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 in a time- and concentration-dependent manner. In contrast, the enzymatic conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 is unaffected by benzylhydrazine. The plot of 1/(inactivation rate) vs 1/[benzylhydrazine] is parabolic, indicating that the inactivation results from the interaction of 2 mol of benzylhydrazine per mole of enzyme. EPR spectra obtained from benzylhydrazine inactivation reactions carried out in the presence of a radical trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, show the formation of a carbon-centered benzyl radical. The benzyl radical most likely results from redox chemistry between benzylhydrazine and the enzyme-bound Cu(II) ions because EPR studies show that enzyme-bound Cu(II) is reduced to Cu(I) in the presence of benzylhydrazine. The kinetic constants for benzylhydrazine as a reductant in the amidation reaction were determined at benzylhydrazine concentrations too low to cause significant enzyme inactivation. Mimosine exhibits mixed inhibition vs benzylhydrazine; however, previous results have shown that benzylhydrazine is competitive vs ascorbate [Miller et al. (1992) Arch. Biochem. Biophys. 298, 380-388]. This change in kinetic mechanism coupled with the nonlinear inactivation kinetics have lead to a proposal that the two enzyme-bound Cu(II) atoms are nonequivalent with respect to their reduction by benzylhydrazine.
Merkler
D J
DJ
Analytical Protein and Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004.
Kulathila
R
R
Ash
D E
DE
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D003300:Copper / Q000737:chemistry*; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D006834:Hydrazines / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008898:Mimosine / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D009097:Multienzyme Complexes
D006834:Hydrazines; D009097:Multienzyme Complexes; D008898:Mimosine; D003300:Copper; C006730:benzylhydrazine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase