Thiorphan, tiopronin, and related analogs as substrates and inhibitors of peptidylglycine α‐amidating monooxygenase (PAM)

McIntyre, Neil; Lowe, Edward W.; Chew, Geoffrey; Owen, Terrence C.; Merkler, David J.

doi:10.1016/j.febslet.2005.12.058

Cited by 17 publications

(12 citation statements)

References 57 publications

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“…The dependence of the initial rate of O 2 consumption on the initial concentration of substrate, at one fixed concentration of ascorbate and O 2 , is well-described by the standard Michealis-Menten equation yielding the steady-state kinetic values for K M,app , V M,app , and (V/K) app included in Table 1. While we have not assayed for glyoxylate or amide formation in each case, previous work on PHM strongly suggests that the binding of the compounds in Table 1 do not simply stimulate the ascorbate-dependent consumption of O 2 , but instead the compounds are oxidized to the amide and glyoxylate 23,24…”

Section: Resultsmentioning

confidence: 97%

“…Since these earlier studies, we23 and others24 have shown that relatively simple N -acyl- and N -arylglycines are PHM substrates with (V/K) app values comparable to the peptide substrates and three dimensional structures of oxidized and reduced PHM have been elucidated to provide a framework for structure-activity data 25. In this report, we further define how simple, substrate-like compounds bind to rat PAM with surprisingly strong affinity.…”

Section: Introductionmentioning

confidence: 99%

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Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine α-hydroxylating monooxygenase (PHM)

Merkler

Asser

Baumgart

et al. 2008

Bioorganic & Medicinal Chemistry

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Peptidyl α-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of α-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C 6 H 5 -CO-NH-CH 2 -COOH) inhibit the enzyme with K i values as low as 0.5 μM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH 2 -COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH 2 -COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K i values. Comparison of K i values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine α-hydroxylating monooxygenase (PHM)

Merkler

Asser

Baumgart

et al. 2008

Bioorganic & Medicinal Chemistry

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“…In addition, tiopronin, a PHM substrate 61 , did protect PHM from inactivation by the cinnamate-mediated inactivation (Figure S4, Supplementary materials). …”

Section: Resultsmentioning

confidence: 98%

“…Copper-free PHM is catalytically inactive 53,62,78 . Cinnamates are known to form complexes with both Cu(I) 79,80 and Cu(II) 81 and copper removal/chelation has been attributed to specific PHM 61 and DβM inhibitors 68 . PHM inactivation requires turnover; thus, inactivation of PHM by cinnamate could result from the formation of a relatively high affinity cinnamate–Cu(I) complex.…”

Section: Discussionmentioning

confidence: 99%

Inactivation of peptidylglycineα-hydroxylating monooxygenase by cinnamic acid analogs

McIntyre

Lowe

Battistini

et al. 2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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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.

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“…Initial velocities were measured at varying concentrations of 2-aminophenol and oxygen. Initial rates were measured by following the tyrosinase-dependent consumption of O 2 using a Yellow Springs Instrument Model 5300 oxygen monitor interfaced with a personal computer using a Dataq Instruments analogue/digital converter (model DI-158RS) interfaced to Microsoft Excel through the Windaq module, modified from McIntyre et al [44]. Using a Maxtec Low flow oxygen blender, in situ [O 2 ] was changed by varying the percent mixtures of oxygen and argon standardized to air-saturated distilled water at 37.0 ± 0.1 °C.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

Washington

Maxwell

Stevenson

et al. 2015

Archives of Biochemistry and Biophysics

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Tyrosinase(EC 1.14.18.1) catalyzes the monophenolase and diphenolase reaction associated with vertebrate pigmentation and fruit/vegetable browning. Tyrosinase is an oxygen-dependent, dicopper enzyme that has three states: Emet, Eoxy, and Edeoxy. The diphenolase activity can be carried out by both the met and the oxy states of the enzyme while neither mono- nor diphenolase activity results from the deoxy state. In this study, the oxidative cyclocondensation of 2-aminophenol (OAP) to the corresponding 2-aminophenoxazin-3-one (APX) by mushroom tyrosinase was investigated. Using a combination of various steady- and pre-steady state methodologies, we have investigated the kinetic and chemical mechanism of this reaction. The kcat for OAP is 75±2 sec−1, KMOAP=1.8±0.20.2emnormalmM, KMO2=25±40.2emnormalμitalicM with substrates binding in a steady-state preferred fashion. Stopped flow and global analysis support a model where OAP preferentially binds to the oxy form over the met (k7 ≫ k1). For the met form, His269 and His61 are the proposed bases, while the oxy form uses the copper-peroxide and His61 for the sequential deprotonation of anilinic and phenolic hydrogens. Solvent KIEs show proton transfer to be increasingly rate limiting for kcat/KMOAP as [O2] −>0 μM (1.38±0.06) decreasing to 0.83±0.03 as [O2]−>∞ reflecting a partially rate limiting μ-OH bond cleavage (Emet) and formation (Eoxy) following protonation in the transition state. The coupling and cyclization reactions of o-quinone imine and OAP pass through a phenyliminocyclohexadione intermediate to APX, forming at a rate of 6.91±0.03 μM−1s−1 and 2.59E-2±5.31E-4 s−1. Differences in reactivity attributed to the anilinic moiety of OAP with o-diphenols are discussed.

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Thiorphan, tiopronin, and related analogs as substrates and inhibitors of peptidylglycine α‐amidating monooxygenase (PAM)

Cited by 17 publications

References 57 publications

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine α-hydroxylating monooxygenase (PHM)

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine α-hydroxylating monooxygenase (PHM)

Inactivation of peptidylglycineα-hydroxylating monooxygenase by cinnamic acid analogs

Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

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