Enzymatic synthesis of radiolabeled phosphonoacetaldehyde.
Phosphonoacetaldehyde (Pald) is formed in a variety of biosynthetic pathways leading to natural phosphonates and is an intermediate in the degradation pathway of the natural product 2-aminoethylphosphonate. To facilitate the investigation of the enzymes catalyzing these pathways, a method for the synthesis of radiolabeled Pald was developed. The enzyme pyruvate phosphate dikinase was used to prepare phosphoenolpyruvate (PEP) from pyruvate, adenosine triphosphate (ATP), and orthophosphate. Then PEP was converted to phosphonopyruvate (Ppyr) with PEP mutase and then to Pald with Ppyr decarboxylase. By using [beta-32P]ATP or [2-14C]pyruvate as precursor, [32P]Pald or [1-14C]Pald was obtained, respectively. The utilization of the synthetic, radiolabeled Pald as a probe of enzyme mechanism was demonstrated with the enzyme phosphonoacetaldehyde hydrolase (trivial name phosphonatase). The single turnover time course for the formation and consumption of radiolabeled covalent enzyme species evidenced a kinetically competent covalent intermediate.
Zhang
Guofeng
G
Department of Chemistry, University of New Mexico, Albuquerque, NM 87131-0001, USA.
Allen
Karen N
KN
Dunaway-Mariano
Debra
D
D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002250:Carbon Radioisotopes / Q000737:chemistry*; D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000737:chemistry*; D017875:Phosphotransferases (Phosphomutases) / Q000737:chemistry / Q000378:metabolism; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D004798:Enzymes; D010761:Phosphorus Radioisotopes; C003126:phosphonoacetaldehyde; D010728:Phosphoenolpyruvate; D011772:Pyruvate, Orthophosphate Dikinase; D006867:Hydrolases; C023920:phosphonoacetaldehyde hydrolase; D002262:Carboxy-Lyases; C113679:PPD protein, Streptomyces viridochromogenes; D017875:Phosphotransferases (Phosphomutases); C057167:phosphoenolpyruvate mutase; D000079:Acetaldehyde