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[52] Citramalate pyruvate lyase
344
REACTIONS LEADING TO AND FROM THE CYCLE
[52]
Divalent Cation Requirement. Ferrous ion is essential in the assay solution to stabilize the activity, even though the system has been activated in the presence of Fe+*. Maximal activity is obtained with 0.2-0.9 mM ferrous ammonium sulfate. Omission or substitution of Fc ++ by Co ++, Ca +*, Cu ++, Mg+*, Zn ++, or Mn ++, in the assay solution results in a rapid decline of activity during the assay. Manganese ion causes the most rapid and extensive inhibition. In the presence of 0.2 mM Fe+*, the same concentration of MnCl, causes a 2 0 ~ inhibition. Equilibrium. The equilibrium constant, K = (L-citramalate)/(mesaconate), at pH 8.4 and 25 ° is between 4.6 and 7.0. A more precise value is not available.
[52] Citramalate Pyruvate Lyase 1
By H. A. BARKER COOH
I
HOC--CH3 --~ CHaCOOH "4- CH3COCOOH
f
CH2
I
COOH Assay Method
Principle. The lyase activity can be measured most conveniently by coupling the above reaction with the reduction of pyruvate by N A D H in presence of excess lactate dehydrogenase. The decrease in absorbance at 340 ms is measured. Reagents Dipotassium L-(~)-citramalate, ~ 0.1 M, or DL-citramalate, 4 0.2M NADH, 1.0 mM MgC12, 10 mM Potassium phosphate buffer, 0.5 M, pH 7.4 Lactate dehydrogenase, about 5 Kornberg units 5 per milliliter I The enzyme has also been called citramalase2 * If. A. Barker, in "The Bacteria" (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. II, p. 151. Academic Press, N e w York, 1961. *H. A. Barker and H. H. Blair, Biochem. Prep. 9, 21 (1962). 'H. A. Barker, Biochem. Prep. 9, 25 (1962). 'A. Kornberg, Vol. I, p. 441.
[52]
CITRAMALATE P Y R U Y A T E LYASE
345
Enzyme: suitably diluted in 10 mM potassium phosphate buffer, pH 7.4 Procedure2 The reaction mixture contains 0.1 M sodium L-(-}-)-citramalate or 0.2 M DL-citramalate, 0.10 ml; 1 mM NADH, 0.10 ml; 10 mM MgC12 0.1 ml; phosphate buffer, 0.1 ml; lactate dehydrogenase solution, 0.02 ml; and lastly, 0.005 to 0.04 unit of citramalate lyase in a total volume of 1.00 ml. The reaction is carried out at 24 ° ~ 1 ° in a 1 ml volume, 1 cm light path silica cuvette. Absorbance readings at 340 m~ are made at 0.5 minute intervals from 0.5 to 2.5 minutes after addition of the enzyme, and the average rate of absorbance change is estimated. During the first 3 minutes the reaction rate remains almost constant. A correction must be applied for N A D H oxidation in the absence of citramalate. Activity is proportional to enzyme concentration over the indicated range. Units. One unit of activity is defined as the amount of enzyme catalyzing the formation of 1 micromole of pyruvate and the oxidation of 1 micromole of N A D H per minute under the assay conditions. This corresponds to a corrected absorbanee change of 6.21 per minute at 340 m#. Specific activity is defined as units per milligram of protein measured by the method of Lowry et al. T using crystalline bovine serum albumin as a standard.
Extraction and Storage of the Enzyme The lyase is obtained from cells of Clostridium tetanomorphum Hl grown anaerobically in a yeast extract-sodium glutamate medium2 Extracts are prepared by treating a cell suspension (15 g of packed cells and 30 ml of distilled water) in a 10 kc Raytheon sonic oscillator for 20 minutes at 0 ° to 5 ° and centrifuging the resulting homogenate for 10 minutes at 11,000 g. The clear supernatant solution (30-35 mg protein per milligram) is either immediately frozen and stored at or below --10 °, or lyophilized and stored at a low temperature as a dry powder. The specific activity of lyophilized preparations ranges from 0.08 to 0.4 unit per milligram, dry weight, or 0.12 to 0.6 unit per milligram of protein. When protected from moisture and stored at --10 °, lyophilized preparations retain activity for more than a year. The specific activity of freshly prepared extracts is 0.8--1.3 units per milligram of protein. The activity of extracts declines rapidly, even at low temperatures. The half-life of • H. A. Barker, Arch. Mi~robiol. 5@, 4 (1967). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). "H. A. Barker, R. D. Smyth, R. M. Wilson, and H. Weisabach, J. Biol. Chem. 234, 320 (1959).
346
REACTIONS LEADING TO AND FROM THE CYCLE
[~2]
lyase activity in 30 mM phosphate buffer pH 7.0 at --15 °, 0 °, 25 °, and 37 ° is about 12 hours, 2.5 hours, 1 hour, and 15 minutes, respectively. Attempts to stabilize the activity in extracts have been unsuccessful. The catalytic properties of the lyase have been determined mainly with unfractionated extracts or with preparations freed of low molecular weight impurities by precipitation with 80% ammonium sulfate, followed by dialysis for a few hours. Properties
Substrate Specificity. L-(+)-Citramalate 9 is the only substrate known to be cleaved by the lyase. D-(--')-Citramalate, DL-isocitrate, DL-malate, citrate, and mesaconate are not converted to keto acid under conditions allowing rapid cleavage of (+)-citramalate to pyruvate and acetate. The apparent Km of sodium L-(~-)-citramalate is 0.6 mM at low substrate concentrations, but it appears to increase at higher substrate concentrations. Maximal activity is obtained with 10-20 mM (-t-)citramalate; at higher concentrations a small inhibition is observed. Effect oJ pH and Buffers. In 50 mM potassium phosphate buffer containing 1 mM MgCI~, the activity-pH curve shows a broad maximum at pH 7.3-7.5. At pH 6.0, the activity is 52%, and at pH 8.0 77% of that at pH 7.4. In 60 mM Tris-HC1 buffer, the activity is maximal at pH 8.0-8.2 and is markedly lower at pH 7.5 and pH 8.8. At pH 7.7, the activity is the same with phosplmte and Tris buffers. EDTA and pyrophosphate buffers are strongly inhibitory. Divalent Cation Requireme~t. A divalent cation, Mg ÷÷, Mn ÷+, or Co÷÷ appears to be essential for activity. The apparent K,~ of MgCl~ is about 0.1 mM. Calcium ion or Fe ÷÷ is unable to substitute for Mg *÷, and these cations at 1 mM inhibit moderately in the presence of 10 #M MgClo. Monovalent cations appear to be inert in this system. Equilibrium. The equilibrium constant for (-t-)-citramalate cleavage at pH 7.4 and 25 ° is about 8.3 M, corresponding to a AF ° value of --1.2 kcal. Occurrence. The enzyme has been observed only in C. tetanornorphum grown with glutamate as a major energy source.
Unpublished experiments of C. C. Wang and H. A. Barker have shown that the citramalate hydro-lyase~° of C. tetanomorphum dehydrates (-~)-citramalate and L-(+)-malate, but not their cnantiomers. Consequently it is probably that the citramalate formed and decomposed by this organism is L-(-b)-citramalate. Z°A. H. Blair and H. A. Barker, J. Biol. Chem. 241, 400 (1966).
REACTIONS LEADING TO AND FROM THE CYCLE
[52]
Divalent Cation Requirement. Ferrous ion is essential in the assay solution to stabilize the activity, even though the system has been activated in the presence of Fe+*. Maximal activity is obtained with 0.2-0.9 mM ferrous ammonium sulfate. Omission or substitution of Fc ++ by Co ++, Ca +*, Cu ++, Mg+*, Zn ++, or Mn ++, in the assay solution results in a rapid decline of activity during the assay. Manganese ion causes the most rapid and extensive inhibition. In the presence of 0.2 mM Fe+*, the same concentration of MnCl, causes a 2 0 ~ inhibition. Equilibrium. The equilibrium constant, K = (L-citramalate)/(mesaconate), at pH 8.4 and 25 ° is between 4.6 and 7.0. A more precise value is not available.
[52] Citramalate Pyruvate Lyase 1
By H. A. BARKER COOH
I
HOC--CH3 --~ CHaCOOH "4- CH3COCOOH
f
CH2
I
COOH Assay Method
Principle. The lyase activity can be measured most conveniently by coupling the above reaction with the reduction of pyruvate by N A D H in presence of excess lactate dehydrogenase. The decrease in absorbance at 340 ms is measured. Reagents Dipotassium L-(~)-citramalate, ~ 0.1 M, or DL-citramalate, 4 0.2M NADH, 1.0 mM MgC12, 10 mM Potassium phosphate buffer, 0.5 M, pH 7.4 Lactate dehydrogenase, about 5 Kornberg units 5 per milliliter I The enzyme has also been called citramalase2 * If. A. Barker, in "The Bacteria" (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. II, p. 151. Academic Press, N e w York, 1961. *H. A. Barker and H. H. Blair, Biochem. Prep. 9, 21 (1962). 'H. A. Barker, Biochem. Prep. 9, 25 (1962). 'A. Kornberg, Vol. I, p. 441.
[52]
CITRAMALATE P Y R U Y A T E LYASE
345
Enzyme: suitably diluted in 10 mM potassium phosphate buffer, pH 7.4 Procedure2 The reaction mixture contains 0.1 M sodium L-(-}-)-citramalate or 0.2 M DL-citramalate, 0.10 ml; 1 mM NADH, 0.10 ml; 10 mM MgC12 0.1 ml; phosphate buffer, 0.1 ml; lactate dehydrogenase solution, 0.02 ml; and lastly, 0.005 to 0.04 unit of citramalate lyase in a total volume of 1.00 ml. The reaction is carried out at 24 ° ~ 1 ° in a 1 ml volume, 1 cm light path silica cuvette. Absorbance readings at 340 m~ are made at 0.5 minute intervals from 0.5 to 2.5 minutes after addition of the enzyme, and the average rate of absorbance change is estimated. During the first 3 minutes the reaction rate remains almost constant. A correction must be applied for N A D H oxidation in the absence of citramalate. Activity is proportional to enzyme concentration over the indicated range. Units. One unit of activity is defined as the amount of enzyme catalyzing the formation of 1 micromole of pyruvate and the oxidation of 1 micromole of N A D H per minute under the assay conditions. This corresponds to a corrected absorbanee change of 6.21 per minute at 340 m#. Specific activity is defined as units per milligram of protein measured by the method of Lowry et al. T using crystalline bovine serum albumin as a standard.
Extraction and Storage of the Enzyme The lyase is obtained from cells of Clostridium tetanomorphum Hl grown anaerobically in a yeast extract-sodium glutamate medium2 Extracts are prepared by treating a cell suspension (15 g of packed cells and 30 ml of distilled water) in a 10 kc Raytheon sonic oscillator for 20 minutes at 0 ° to 5 ° and centrifuging the resulting homogenate for 10 minutes at 11,000 g. The clear supernatant solution (30-35 mg protein per milligram) is either immediately frozen and stored at or below --10 °, or lyophilized and stored at a low temperature as a dry powder. The specific activity of lyophilized preparations ranges from 0.08 to 0.4 unit per milligram, dry weight, or 0.12 to 0.6 unit per milligram of protein. When protected from moisture and stored at --10 °, lyophilized preparations retain activity for more than a year. The specific activity of freshly prepared extracts is 0.8--1.3 units per milligram of protein. The activity of extracts declines rapidly, even at low temperatures. The half-life of • H. A. Barker, Arch. Mi~robiol. 5@, 4 (1967). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). "H. A. Barker, R. D. Smyth, R. M. Wilson, and H. Weisabach, J. Biol. Chem. 234, 320 (1959).
346
REACTIONS LEADING TO AND FROM THE CYCLE
[~2]
lyase activity in 30 mM phosphate buffer pH 7.0 at --15 °, 0 °, 25 °, and 37 ° is about 12 hours, 2.5 hours, 1 hour, and 15 minutes, respectively. Attempts to stabilize the activity in extracts have been unsuccessful. The catalytic properties of the lyase have been determined mainly with unfractionated extracts or with preparations freed of low molecular weight impurities by precipitation with 80% ammonium sulfate, followed by dialysis for a few hours. Properties
Substrate Specificity. L-(+)-Citramalate 9 is the only substrate known to be cleaved by the lyase. D-(--')-Citramalate, DL-isocitrate, DL-malate, citrate, and mesaconate are not converted to keto acid under conditions allowing rapid cleavage of (+)-citramalate to pyruvate and acetate. The apparent Km of sodium L-(~-)-citramalate is 0.6 mM at low substrate concentrations, but it appears to increase at higher substrate concentrations. Maximal activity is obtained with 10-20 mM (-t-)citramalate; at higher concentrations a small inhibition is observed. Effect oJ pH and Buffers. In 50 mM potassium phosphate buffer containing 1 mM MgCI~, the activity-pH curve shows a broad maximum at pH 7.3-7.5. At pH 6.0, the activity is 52%, and at pH 8.0 77% of that at pH 7.4. In 60 mM Tris-HC1 buffer, the activity is maximal at pH 8.0-8.2 and is markedly lower at pH 7.5 and pH 8.8. At pH 7.7, the activity is the same with phosplmte and Tris buffers. EDTA and pyrophosphate buffers are strongly inhibitory. Divalent Cation Requireme~t. A divalent cation, Mg ÷÷, Mn ÷+, or Co÷÷ appears to be essential for activity. The apparent K,~ of MgCl~ is about 0.1 mM. Calcium ion or Fe ÷÷ is unable to substitute for Mg *÷, and these cations at 1 mM inhibit moderately in the presence of 10 #M MgClo. Monovalent cations appear to be inert in this system. Equilibrium. The equilibrium constant for (-t-)-citramalate cleavage at pH 7.4 and 25 ° is about 8.3 M, corresponding to a AF ° value of --1.2 kcal. Occurrence. The enzyme has been observed only in C. tetanornorphum grown with glutamate as a major energy source.
Unpublished experiments of C. C. Wang and H. A. Barker have shown that the citramalate hydro-lyase~° of C. tetanomorphum dehydrates (-~)-citramalate and L-(+)-malate, but not their cnantiomers. Consequently it is probably that the citramalate formed and decomposed by this organism is L-(-b)-citramalate. Z°A. H. Blair and H. A. Barker, J. Biol. Chem. 241, 400 (1966).