scholarly journals NN-dimethyl-1,4-phenylenediamine as an alternative reductant for peptidylglycine α-amidating mono-oxygenase catalysis

1994 ◽  
Vol 300 (1) ◽  
pp. 31-36 ◽  
Author(s):  
C Li ◽  
C D Oldham ◽  
S W May
Keyword(s):  
Cation Radical ◽  
Kinetic Studies ◽  
Peptide Hormone ◽  
Steady State Kinetic ◽  
Rate Of Increase ◽  
Ping Pong ◽  
Assay Methods ◽  
Menten Kinetic ◽  
Continuous Assay ◽  
State Kinetic

C-terminal alpha-amidation is a structural feature essential to the biological activity of many peptide hormones. Peptidylglycine alpha-amidating mono-oxygenase (PAM; EC 1.14.17.3) catalyses conversion of glycine-extended peptide hormone precursors into their corresponding alpha-hydroxyglycine derivatives. This reaction is the first step in the C-terminal amidation process. We report here that in the presence of molecular O2, copper and PAM substrate, NN-dimethyl-1,4-phenylenediamine (DMPD) serves as the requisite electron donor for the mono-oxygenase, being oxidized in the process to a stable and highly chromophoric cation radical. By monitoring the rate of increase in absorbance at 515 nm, PAM activity can be easily followed. This provides a spectrophotometric assay for PAM, which represents the first continuous assay reported for this enzyme. DMPD-supported PAM-catalysed mono-oxygenation exhibits normal Michaelis-Menten kinetic behaviour. Steady-state kinetic studies established that both the ascorbate-supported and DMPD-supported PAM reactions exhibit apparent ‘Ping Pong’ kinetics. In addition, both electron donors give rise to similar pH profiles and identical inhibition patterns towards known competitive inhibitors of PAM. The stoichiometry between formation of the DMPD cation radical and the alpha-hydroxyglycine PAM product was determined to be 2:1, the value expected for a monooxygenase-catalysed reaction. The optimum pH for the DMPD-supported continuous PAM assay was found to be about 5.5. The major advantage of this assay over all previously reported methods is that it is continuous; thus accurate initial rates are easily obtained. Moreover, unlike previous assay methods, 125I-labelled or chromophorically modified substrates are not required. Kinetic parameters for a broad range of PAM substrates and inhibitors have been successfully obtained using this assay.

Pre-steady-state kinetic studies of rat kidneyγ-glutamyl transpeptidase confirm its ping-pong mechanism

2004 ◽  
Vol 17 (67) ◽  
pp. 529-536 ◽  
Author(s):  
Jeffrey W. Keillor ◽  
Annie Ménard ◽  
Roselyne Castonguay ◽  
Christian Lherbet ◽  
Caroline Rivard
Keyword(s):  
Steady State ◽  
Kinetic Studies ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Glutamyl Transpeptidase ◽  
State Kinetic

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

2001 ◽  
Vol 360 (3) ◽  
pp. 727-736 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Christian EIS
Keyword(s):  
Steady State ◽  
Transition State ◽  
Schizophyllum Commune ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

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