Abstract
Carbonyl sulfide, a competitive inhibitor of ribulose-bisphosphate carboxylase with respect to CO2 (Laing, W. A., and Christeller, J. T. (1980) Arch. Biochem. Biophys. 202, 592-600), is an alternate substrate. Thiocarboxylation was monitored by mass spectrometry as the stoichiometric consumption of carbonyl sulfide. The product, 1-thio-3-phosphoglycerate, was identified by 13C NMR and UV absorption spectroscopy and measured by enzymic conversion to thiolactate, coupled to the oxidation of NADH. The expected stoichiometry of thiocarboxylation was confirmed. The maximal rates of thiocarboxylation for the spinach and Rhodospirillum rubrum enzymes were close to the maximal rates of carboxylation for these two enzymes. Both enzymes favored CO2 over carbonyl sulfide (with Mg2+ as metal ion) by a factor of about 110. Thiocarboxylation could only be demonstrated with enzymes carbamylated with CO2. Incubation of the carbamylated E.ACO2.Mg complex with excess carbonyl sulfide caused the displacement of the activator carbamate. The thiocarbamylated enzyme was catalytically incompetent and did not form a stable quaternary complex with 2'-carboxyarabinitol bisphosphate. Incubation of the thiocarbamylated enzyme with excess CO2 resulted in the displacement of the thiocarbamate, the re-formation of the carbamylated E.ACO2.Mg complex and the restoration of catalytic competence. Computergraphic simulation of the thiocarbamylated quaternary complex indicated unfavorable van der Waals interactions associated with the thiocarbamate.
Highlights
From the Central Research and Development Department,E
The enzyme is first activated by reacting with a molecule of CO, which is distinctfrom the molecule of COz which is subsequently fixed during catalysis (Miziorko, 1979; Lorimer, 1979).*C02’ reactswiththe tamino group of Lys-201 of the large subunit to form a carbamate residue (Lorimerand Miziorko, 1980; Lorimer, 1981) which is in close proximity to a catalytically essential divalent dead-endinhibitor competitivewithrespect to the second substrate would be expected to be uncompetitive with respect to the first substrate, Laing and Christel(l1e9r 80) concluded that the carboxylase reaction was random
The kinetic order of the carboxylase reaction has been the subject of conflicting reports(Laing and Christeller, 1980; Badger et al, 1980; Roeske and O’Leary, 1984,1985; Pierce et u., 1986a; Van Dyk and Schloss, 1986).The most compelling evidence for a random mechanism came from the studies of Laing and Christeller (1980) on the pattern of inhibition of the enzyme by COS
Summary
From the Central Research and Development Department,E. I. The enzyme is first activated by reacting with a molecule of CO, which is distinctfrom the molecule of COz which is subsequently fixed during catalysis (Miziorko, 1979; Lorimer, 1979).*C02’ reactswiththe tamino group of Lys-201 of the large subunit to form a carbamate residue (Lorimerand Miziorko, 1980; Lorimer, 1981) which is in close proximity to a catalytically essential divalent dead-endinhibitor competitivewithrespect to the second substrate would be expected to be uncompetitive with respect to the first substrate, Laing and Christel(l1e9r 80) concluded that the carboxylase reaction was random This conclusion rests critically upon the assumption thatCOS is a true deadend inhibitor and not an alternative substrate. Sinwcee have reached the conclusion that thecarboxylase reaction is ordered with ribulose-Pz binding first (Pierceet al., 1986a), it behove us to reexamine the question as to whether or not
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