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J. Biol. Chem., Vol. 261, Issue 22, 10248-10256, Aug, 1986

Reaction intermediate partitioning by ribulose-bisphosphate carboxylases with differing substrate specificities

J Pierce, TJ Andrews and GH Lorimer

The carboxylated, 6-carbon reaction intermediate (3-keto-2- carboxyarabinitol 1,5-bisphosphate) from the ribulose-1,5-bisphosphate carboxylase reaction was obtained by denaturing the enzyme with acid during steady-state turnover. Carbon-13 NMR analysis indicates that this beta-keto acid exists in solution predominantly as the C-3 ketone (as opposed to the hydrate) form. In neutral solution the intermediate slowly decomposes (t1/2 approximately 1 h) by decarboxylation. This decarboxylation reaction is catalyzed by nonactivated (metal free) ribulose-1,5-bisphosphate carboxylase. Alternately, the activated enzyme predominantly catalyzes the hydrolysis of the intermediate to two molecules of glycerate 3-phosphate. The partitioning of the intermediate (i.e. hydrolysis/(hydrolysis + decarboxylation] by activated ribulose-1,5-bisphosphate carboxylase was studied using enzymes from three different sources and with different activating metal atoms. This afforded a series of catalysts whose relative specificities for the alternate substrates, carbon dioxide and oxygen, varied over a 50-fold range. When Mg2+ was the activating metal, the partitioning of the reaction intermediate varied only from 0.93 to 1 for all three enzymes. Even the Co2+ activated enzyme from Rhodospirillum rubrum, which is completely devoid of carboxylase activity, partitioned approximately 30% of added intermediate to products. It is probable that the 6-carbon intermediate's strong commitment to product formation is paralleled by a similarly strong forward commitment of the analogous intermediate in the oxygenase reaction. In this event, the variations in relative specificity for the gaseous substrates of enzymes from different natural sources must arise by interactions that take place on the enzyme prior to the formation of the intermediates.
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