Abstract
The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.
Highlights
[phenazine methosulfate (PMS)] plots, at various constant concentrations of a reducing substrate,are parallel
The electron acceptor for several bacterial methylamine dehydrogenases is the copper protein amicyanin [7,8,9,10,11], a soluble C-type cytochrome has been implicated as the electron acceptor for the methylamine dehydrogenases from bacterium W3A1 [12]
Notable are: the EPR and electron nuclear double amine or phenazine ethosulfate (PES) is dependent on the ionic strength, and resonance spectrometry studies on glucose, methanol, and the data indicate that each interacts with an anionic methylamine dehydrogenase [14,15,16]; studies on methanol site on methylamine dehydrogenase
Summary
Ylammonium, ethylammonium, propylammonium, and butylammonium ions, respectively; PES and PMS, phenazine ethosulfate and phenazine methosulfate, respectively; Ps.,Pseudomonas; SSQ, sum Materials Bacterium W3A1wasgrown as previously reported [26]except of the squared residuals. BuNHz and ethanolamine as well as the hydrochlorides of MeNH2,EtNH2, PrNH2, and1,3-diaminopropane were purchased from Aldrich. BuNH2 and ethanolamine were converted to the hydrochlorides by bubbling dry HC1 gas through an ethersolution of these amines. MeNH3Cl and EtNH3Cl weretwice recrystallized from methanol, and the other amine hydrochlorides were recrystallized from an ether/ethanol mixture. Horse heart cytochrome c (type VI), phenazine methosulfate (PMS), phenazine ethosulfate (PES), N,N,N’,N’-tetramethyl-p-phenylenediamine,a-ketoglutarate, NADH, and glutamate dehydrogenase (type 11) were purchased from Sigma. 2,6-Dichlorophenolindophenolwas supplied by General Biochemicals, Inc. Cytochrome CM,~ from Pseudomonas stutzerii was a kind gift from Dr T. Wurster’s blue, the radical of N,N,N‘,N’-tetramethyl-p-phenylenediaminew,as synthesized as described earlier [27]
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