The Mechanism of Action of Carboxydismutase

Carboxydismutase is inhibited by iodoacetanide, and the substrate RuDP protects the enzyme against this inhibition.' The magnitude of the rate of inhibition and the variation of this rate with pH suggested that the mercaptide form of a cysteine residue was alkylated by the inhibitor. A nmechanisn was proposed in which this mercaptide reacted with the carbonyl group of RuDP to give a thiohemiacetal. It was suggested that water was eliminated from this compound to give an enol which then reacted with enzyme-bound bicarbonate. Further evidence relevant to these proposals has now been obtained by spectrophotometric investigations and by use of the sulphydryl reagent DTNB. 2 Experimental.-Enzyme: This was prepared and assayed as previously described.l Buffers: These were prepared from Trizma (Sigma Chemical Co., St. Louis) and redistilled hydrochloric acid. A stock solution (total tris, 1 M, pH 8.0) was freed from metal ions as previously described.' RuDP: RuDP was prepared and purified by the method of Rabin et al.3 lodoacetamide 1-Ct4: This was purchased from Tracerlab (Waltham, Mass. ) and was stated to have a specific activity of 1.25 mC/mM. DTNB: This was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin. A stock solution (10 mM, pH 7.2) was prepared as follows. DTNB (39.6 mg, 0.1 mmole) was dissolved in 10 ml of a solution containing tris base (0.2 mmole) and tris/HCl buffer, (pH 7.2, 0.5 mmole with respect to total tris). The solution was centrifuged (500 X g for 10 min) before use. The stock was diluted with water to give a I mM solution. Difference spectrophotometry: All measurements were made with a Cary Model 14 spectrophotometer with a 0-0.1, 0.1-0.2 slide wire. Split-compartment cells, as described by Yankeelov,4 but with a total light path of 1.0 cm, were employed. These cells were manufactured to special order by Pyrocell Manufacturing Co., New York, New York. A solution of carboxydismutase in tris buffer (1 ml) was pipetted into the front compartment of both the reference and sample cells. A solution of RuDP in the same buffer (1 ml) was then pipetted into the rear compartments of the cells, and the base line recorded. The contents of the sample cell were then mixed by inversion, and the difference spectrum was recorded. Subsequently, the reference cell was inverted and the base line checked. Invariably no change in the base line was observed. Reaction with DTNB: The reaction was started by the addition of DTNB solution (50 ul, 1 or 10 mnM) to the enzyme in 0.95-ml solution containing tris buffer pH 8.0 to give a final concentration of 0.05 M with respect to total tris. The blank cell contained the same concentration of DTNB and buffer, and both cells had a total optical path of I cm. The reaction was followed by observing the change in absorbance at 410 mu, using a Cary recording spectrophotometer. The molar extinction coefficient of the chromophore produced in the reaction with cysteine under these conditions was 14,000. Beer's law was found to be obeyed over the absorbance range observed in the enzyme experiments. Preparation of alkylated enzyme: Carboxydismutase (2.7 ml, 3 mg/ml) was incubated in 0.4 AM tris buffer (pH 8.0) with iodoacetamide (1 mM, final concentration) at 25? for 45 min. The solution was cooled to 0? and dialyzed by the method of Hospelhorn' against 0.4 M tris buffer (pH 8.0) at 2? for 3 hr. It was used in the spectrophotometric experiments without further purification. Isolation and identification of Ca4-carboxymethylcysteine from alkylated carhoxydismutase: Carboxydismutase (2 ml, 5 mg/ml) in tris buffer (0.064 M, pH 8.0) was treated with iodoacetamide 1-C14 (0.37 mg, 2 uM) at 25? for 30 min. The reaction mixture was cooled to 0?, and dialyzed by the method of Hospelhorn5 against deionized water taken to pH 8.0 by the addition of ammonia. After 1.6 1 of dialyzing solution had flowed over the sac, the dialysate had no detectable radioactivity. The protein solution was freeze-dried, dissolved in redistilled HCI (6.0 N), deaerated, and 88