Abstract
Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.
Acknowledgements
The authors acknowledge fruitful discussions of this work with Dr. Randy W. Larsen and technical support from Jason Cuce.
Declaration of interest
This work was supported, in part, by grants from the National Institutes of Health (R15-GM073659 and R03-DA03432), the Gustavus and Louise Pfieffer Research Foundation, the University of South Florida – Established Researcher Grant Program, the Wendy Will Case Cancer Fund, the Eppley Foundation for Research, the Milheim Foundation for Cancer Research, the Shin Foundation for Medical Research, and the Shirley W. & William L. Griffin Foundation to D.J.M.; a predoctoral fellowship from the American Heart Association (0415259B) and a postdoctoral fellowship from the National Science Foundation CI-TraCS Program (OCI-1122919) to E.W.L.; and from the National Institutes of Health (NIH-RCMI, 2G12MD007595-04 and 1SC3GM112558-01) and Louisiana Cancer Research Consortium to N.R.M. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the Louisiana Cancer Research Consortium or the National Institutes of Health.
Supplementary material available online
Supplementary Figures S1-S5.
Notes
*A reviewer noted that the no cinnamate control in exhibited loss of activity. A repeat of the experiment to eliminate the activity loss in the no cinnamate control is difficult to accomplish because PHM exhibits a loss of activity under turnover conditions, probably the result of reactive, reduced O2-species that from during catalysis (see referenceCitation77). The inclusion of catalase in the inactivation experiments serves to decrease this problem as much as possible.
†The inactivation and inhibition parameters were measured at one fixed concentration of O2 and ascorbate and are, thus, reported as the kinact,obs, KI,obs, and (kinact/KI)obs.