L-phenylalanine ammonia-lyase (maize, potato, and Rhodotorula glutinis). Studies of the prosthetic group with nitromethane.
Highly purified enzyme (EC 4.1.3.5) from Rhodotorula glutinis was shown by sodium dodecyl sulfate gel electrophoresis to have subunits which if not identical are closely similar in molecular weight. Like the enzyme from maize and potato [Havir, E. A., and Hanson, K. R. (1973), Biochemistry 12, 1583] it is a tetramer of molecular weight similar to 4 times 83,000. Enzyme from all three sources inactivated and labeled at the active site with 14-CH3NO2 gave on HCl hydrolysis 14-CO2, H-14-CO2H, D- and L-[14-C]aspartic acid, and unidentified radioactive products. In addition, the labeled R. glutinis enzyme gave [1,2-14-C2]glycine. The formation of the first three products is compatible with the hypothesis that the electrophilic prosthetic group of the enzyme contains the dehydroalanine imine system greater than C equals to N minus C-alpha(equals to C-beta-H2)COminus and inactivation involves attack on C-beta. The second-order rate constants for CH3NO2 inactivation varied with pH as a simple titration curve. The pKa values calculated from the curves for the three enzymes differed and were lower than the pKa of CH3NO2 by at least 1 pH unit. Apparently the inactivation process is enzyme catalyzed. Both inactivation and addition of the substrate amino group may occur with attack on C-beta.
D000642:Ammonia-Lyases / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008697:Methane; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D009574:Nitro Compounds; D010649:Phenylalanine; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D012248:Rhodotorula / Q000201:enzymology*; D013045:Species Specificity; D003313:Zea mays / Q000201:enzymology
D046911:Macromolecular Substances; D009574:Nitro Compounds; D010649:Phenylalanine; D000642:Ammonia-Lyases; D008697:Methane