Identification of reaction products and intermediates of aromatic-amine dehydrogenase by 15N and 13C NMR.
13C- and 15N-NMR studies of the reaction of aromatic amine dehydrogenase (AADH) with methylamine demonstrated that the products of the reductive half-reaction are an equivalent of formaldehyde hydrate and a reduced aminoquinol form of the tryptophan tryptophylquinone (TTQ) cofactor which contains covalently bound substrate-derived N. These data are consistent with the Ping Pong kinetic mechanism and aminotransferase-type chemical reaction mechanism which have been previously proposed for AADH. Comparison of the 15N-NMR spectra of the aminoquinol TTQ intermediates of AADH and methylamine dehydrogenase (MADH) revealed that the substrate-derived aminoquinol N of AADH and MADH exhibited distinct 15N chemical shifts which are separated by approx. 7 p.p.m. In each case, the signal for the substrate-derived aminoquinol N appears optimally with short pulse delay and exhibits a relaxation time and chemical shift which are consistent with 15N covalently bound to an aromatic ring (i.e. aminoquinol) which is attached to a rigid protein matrix. The aminoquinol of AADH is less stable against reoxidation than that of MADH. These data suggest that differences in the active-site mediated electrostatic environments of the aminoquinol N in the respective enzymes may influence both the observed 15N chemical shift and the relative reactivities of the TTQ aminoquinols towards oxygen. These data also demonstrate the utility of 13C- and 15N-NMR spectroscopy as a tool for monitoring the intermediates and products of enzyme-catalysed transformations.
Bishop
G R
GR
Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216-4505, USA.
Zhu
Z
Z
Whitehead
T L
TL
Hicks
R P
RP
Davidson
V L
VL
D000421:Alcaligenes / Q000201:enzymology; D002247:Carbon Isotopes; D002384:Catalysis; D004795:Enzyme Stability; D005583:Fourier Analysis; D007700:Kinetics; D008744:Methylamines / Q000737:chemistry / Q000378:metabolism*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D002247:Carbon Isotopes; D008744:Methylamines; D009587:Nitrogen Isotopes; C027451:methylamine; D000587:Oxidoreductases Acting on CH-NH Group Donors; C037193:aromatic amine dehydrogenase