title: Intermediates in the ribulose-1,5-bisphosphate carboxylase reaction.
abstract: At least two intermediates of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) reaction were liberated in detectable amounts when the functioning enzyme from Rhodospirillum rubrum was quenched in acid. Using substrate labeled with 32P in C-1, [32P]orthophosphate (Pi) was found when the quenched solution was rapidly processed for extraction of Pi as the acid molybdate complex. Reaction with sodium borohydride under mildly alkaline conditions immediately after acid quenching of the carboxylase reaction decreased the amount of 32Pi that was observed by 68%. The compound whose degradation to Pi was prevented by reaction with sodium borohydride decomposed under both acid and neutral conditions with a half-time of about 5 min at 25 degrees C and was assigned to the beta-keto acid recently demonstrated for the spinach enzyme ( Schloss , J.V., and Lorimer , G.H. (1982) J. Biol. Chem. 257, 4691-4694). It was sufficiently stable upon neutralization to react productively with fresh enzyme. As substrate CO2 concentration was decreased below the steady state Km value, the proportion of the 32P that did not react with sodium borohydride increased, indicative of a second unstable intermediate that precedes the carboxylation step. The decomposition of the latter intermediate to Pi, which occurs with a t1/2 less than or equal to 6 ms, was prevented if I2 was present in the acid quench medium. These are properties expected of the 2,3- enediol form of ribulose bisphosphate. Both intermediates reach their maximum levels when product formation is most rapid and disappear when product formation is complete as expected of reaction intermediates.
mesh_terms: D001894:Borohydrides / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D007455:Iodine / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D012274:Ribulosephosphates / Q000378:metabolism
chemical_list: D001894:Borohydrides; D010710:Phosphates; D012274:Ribulosephosphates; D002245:Carbon Dioxide; C001933:ribulose-1,5 diphosphate; C025364:sodium borohydride; D007455:Iodine; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen