Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1.
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.
D000588:Amines / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology*; D002082:Butylamines / Q000378:metabolism; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D008773:Methylphenazonium Methosulfate / Q000378:metabolism; D009994:Osmolar Concentration; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010619:Phenazines / Q000378:metabolism; D011437:Propylamines / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*
D000588:Amines; D002082:Butylamines; D008744:Methylamines; D010619:Phenazines; D011437:Propylamines; D000644:Quaternary Ammonium Compounds; C028122:5-ethylphenazine; D008773:Methylphenazonium Methosulfate; C027451:methylamine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors