13C kinetic isotope effects on the reaction of a flavin amine oxidase determined from whole molecule isotope effects.
A large number of flavoproteins catalyze the oxidation of amines. Because of the importance of these enzymes in metabolism, their mechanisms have previously been studied using deuterium, nitrogen, and solvent isotope effects. While these results have been valuable for computational studies to distinguish among proposed mechanisms, a measure of the change at the reacting carbon has been lacking. We describe here the measurement of a13C kinetic isotope effect for a representative amine oxidase, polyamine oxidase. The isotope effect was determined by analysis of the isotopic composition of the unlabeled substrate, N, N'-dibenzyl-1,4-diaminopropane, to obtain a pH-independent value of 1.025. The availability of a13C isotope effect for flavoprotein-catalyzed amine oxidation provides the first measure of the change in bond order at the carbon involved in this carbon-hydrogen bond cleavage and will be of value to understanding the transition state structure for this class of enzymes.
Department of Chemistry and Biochemistry, St. Mary's University, San Antonio, TX 78228, United States.
José R
JR
Tormos
Department of Geological Sciences, University of Texas-San Antonio, San Antonio, TX 78249, United States.
Marina B
MB
Suarez
Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, United States. Electronic address: fitzpatrickp@uthscsa.edu.
Paul F
PF
Fitzpatrick
D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005415:Flavins / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008995:Monoamine Oxidase / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D013696:Temperature; D000097667:Polyamine Oxidase
D002247:Carbon Isotopes; D005415:Flavins; D005420:Flavoproteins; D008995:Monoamine Oxidase; D010088:Oxidoreductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011073:Polyamines; D000097667:Polyamine Oxidase
Dehydrogenase; Enzyme mechanisms; Flavoprotein; Polyamine oxidase