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[122] Lactyl-CoA dehydrase
[122]
LACTVL-COA DEHYDRASE
683
[ 122 ] L a c t y l - C o A D e h y d r a s e
By R. L. BALDWIN and W. k. WOOD Acrylyl-CoA+ H20 ~ lactyl-CoA Assay Method 1
Principle. The enzyme can be assayed spectrophotometrically with acrylyl-CoA as substrate. The rate of lactyl-CoA formation is determined continuously by conversion of this product to free lactate in the presence of acetate and CoA transphorase. The lactate is then oxidized with a muscle lactic dehydrogenase, NAD, diaphorase dye system. The rate of reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl) tetrazolium chloride (INT) is determined at 500 m#. Reagents Tris-aeetate buffer, 0.2 M, pH 8.0 INT, 0.2% solution NAD, 0.15 M Acrylyl-CoA, ~ 0.005 M Muscle lactic dehydrogenase Coenzyme A transphorase x Diaphorase Procedure. The assay mixture contains Tris-acetate buffer, pH 8.0, 20 micromoles; INT, 20 ~g; NAD, 1.5 micromoles; acrylyl-CoA, 0.1 micromole; muscle lactic dehydrogenase, 0.5 unit; diaphorase, 1.0 unit; CoA transphorase, 1.0 unit, and lactyl-CoA dehydrase in a final volume of 0.30 ml. Either lactyl-CoA dehydrase or acrylyl-CoA can be added last. Often in crude enzyme preparations, it is desirable to preincubate the assay mixture without acrylyl-CoA for several minutes until the endogenous rate of dye reduction decreases. Coenzyme A transphorase and diaphorase from Peptostreptococcus elsdenii are fairly heat stable whereas lactyl-CoA dehydrase is not; hence, heated (55 ° for 10 minutes) crude extracts of P. elsdenii can replace purified CoA transphorase and diaphorase in the assay system. The reaction proceeds linearly with time for 4-6 minutes and is linear with enzyme concentration when the reaction rate is below 0.025 absorbancy unit per minute. Crude extracts contain very high levels of crotonase and fl-hydroxybutyryloCoA dehyI R. L. Baldwin, W. A. Wood, and R. S. Emery, Biochim. Biophys. Acta 97, 202 (1965).
2See Vol. III, p. 931.
684
DEHYDRASES
[122]
drogenase, which can reduce I N T in the presence of acrylyl-CoA. Hence, in crude extracts it is essential that only the activity requiring lactic dehydrogenase for expression is considered. Definition o] Unit and Specific Activity. One unit of enzyme will catalyze the formation of 1.0 ~mole of laetyl-CoA per minute at 25 °, and specific activity is expressed as units per milligram of protein. The millimolar extinction coefficient for I N T was determined to be 20.4 X 103. This value is somewhat higher than reported by Hirsch et al. 3 but similar to that reported by Penningten 4 for I N T in a nonaqueous system. Purification Proce4ure
Preparation o] Crude Extract. P. elsdenii, strain B 159,5 was grown on 1 liter of medium containing 1% corn steep liquor, 1.4% sodium DLlactate, and 0.02% mercaptoethanol (see Walker 6 for details of the procedure). After growth at 38 °, the cells were collected by centrifugation, washed twice with cold 0.02 M potassium phosphate buffer (pH 7.5), and disrupted by sonic oscillation. The suspension of disrupted cells was centrifuged at 20,000 g for 30 minutes to remove cellular debris. The crude extracts thus obtained were stored at --15°C. Nucleic Acid Precipitation. The protein concentration of the crude extract was adjusted to approximately 15 mg/ml with 0.05 M potassium phosphate buffer (pH 7.5), and solid ammonium sulfate was added to a final concentration of 0.20 M. The nucleic acids were then precipitated by the dropwide addition of 0.20 volume of 1.0% protamine. The enzyme is easily precipitated with protamine and, hence, this step must be run carefully. First Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to the supernatant from the protamine step to 50% saturation, centrifuged, and the precipitate discarded. The supernatant was adjusted to 65% saturation with solid ammonium sulfate, centrifuged, and the precipitate dissolved and adjusted to twice the original volume with water. Fractionation with Calcium Phosphate Gel. Calcium phosphate gel was added in a stepwise fashion until about 80% of the enzyme was adsorbed. The gel was then washed with 0.07 M phosphate buffer, pH 7.5, and then the enzyme was eluted from the gel by washing twice with 3C. A. Hirsch, M. Rasminsky, B. D. Davis, and E. C. C. Lin, J. Biol. Chem. 3770 (1963). ' R. J. Pennington, Biochem. J. 80, 649 (1961). 5Obtained from Dr. Marvin Bryant, Department of Dairy Science, University of Illinois, Urbana, Illinois. eD. J'. Walker, Biochem. J. 69, 524 (1958).
[122]
LACTYL-COA DEHYDRASE
685
volumes of 0.12M phosphate buffer, pH 7.5, equal to about twice the volume of the gel. Second Ammonium Sul]ate Fractionation. The eluates from the calcium phosphate gel were combined and solid ammonium sulfate added to a final concentration of 55% saturation. The precipitate was discarded and the ammonium sulfate concentration adjusted to 70% saturation. The precipitate was collected and resuspended in 0.5 M phosphate buffer, pH 7.5. A summary of the procedure is shown in the accompanying table. PURIFICATION PROCEDURE
Fraction Crude extract Protamine First ammonium sulfate Calcium phosphate gel Second ammonium sulfate
Total Volume activity (ml) (units) 15 35 60 17 10
2.76 2.58 2.20 1.50 1.10
Specific activity (units/mg Yield Protein protein (%) (mg) X 10t) 93 80 54 40
480 420 126 37 16
O. 58 O. 61 1.75 4.05 6.90
Properties The enzyme has not been purified sufficiently to study in detail the kinetics of the reaction and the effects of inhibitors and activators. The pH optimum for the reaction appears to be between 7.5 and 8.0. The activity observed with crotonyl-CoA as substrate is 75% that observed with acrylyl-CoA. EDTA does not inhibit, and sometimes stimulates, activity. The enzyme is relatively stable in crude extracts stored at --15 ° . However, it becomes very unstable during purification. For example, approximately 10% of the enzyme activity is lost per hour when the enzyme is allowed to stand at 0 ° after the first ammonium sulfate step.
LACTVL-COA DEHYDRASE
683
[ 122 ] L a c t y l - C o A D e h y d r a s e
By R. L. BALDWIN and W. k. WOOD Acrylyl-CoA+ H20 ~ lactyl-CoA Assay Method 1
Principle. The enzyme can be assayed spectrophotometrically with acrylyl-CoA as substrate. The rate of lactyl-CoA formation is determined continuously by conversion of this product to free lactate in the presence of acetate and CoA transphorase. The lactate is then oxidized with a muscle lactic dehydrogenase, NAD, diaphorase dye system. The rate of reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl) tetrazolium chloride (INT) is determined at 500 m#. Reagents Tris-aeetate buffer, 0.2 M, pH 8.0 INT, 0.2% solution NAD, 0.15 M Acrylyl-CoA, ~ 0.005 M Muscle lactic dehydrogenase Coenzyme A transphorase x Diaphorase Procedure. The assay mixture contains Tris-acetate buffer, pH 8.0, 20 micromoles; INT, 20 ~g; NAD, 1.5 micromoles; acrylyl-CoA, 0.1 micromole; muscle lactic dehydrogenase, 0.5 unit; diaphorase, 1.0 unit; CoA transphorase, 1.0 unit, and lactyl-CoA dehydrase in a final volume of 0.30 ml. Either lactyl-CoA dehydrase or acrylyl-CoA can be added last. Often in crude enzyme preparations, it is desirable to preincubate the assay mixture without acrylyl-CoA for several minutes until the endogenous rate of dye reduction decreases. Coenzyme A transphorase and diaphorase from Peptostreptococcus elsdenii are fairly heat stable whereas lactyl-CoA dehydrase is not; hence, heated (55 ° for 10 minutes) crude extracts of P. elsdenii can replace purified CoA transphorase and diaphorase in the assay system. The reaction proceeds linearly with time for 4-6 minutes and is linear with enzyme concentration when the reaction rate is below 0.025 absorbancy unit per minute. Crude extracts contain very high levels of crotonase and fl-hydroxybutyryloCoA dehyI R. L. Baldwin, W. A. Wood, and R. S. Emery, Biochim. Biophys. Acta 97, 202 (1965).
2See Vol. III, p. 931.
684
DEHYDRASES
[122]
drogenase, which can reduce I N T in the presence of acrylyl-CoA. Hence, in crude extracts it is essential that only the activity requiring lactic dehydrogenase for expression is considered. Definition o] Unit and Specific Activity. One unit of enzyme will catalyze the formation of 1.0 ~mole of laetyl-CoA per minute at 25 °, and specific activity is expressed as units per milligram of protein. The millimolar extinction coefficient for I N T was determined to be 20.4 X 103. This value is somewhat higher than reported by Hirsch et al. 3 but similar to that reported by Penningten 4 for I N T in a nonaqueous system. Purification Proce4ure
Preparation o] Crude Extract. P. elsdenii, strain B 159,5 was grown on 1 liter of medium containing 1% corn steep liquor, 1.4% sodium DLlactate, and 0.02% mercaptoethanol (see Walker 6 for details of the procedure). After growth at 38 °, the cells were collected by centrifugation, washed twice with cold 0.02 M potassium phosphate buffer (pH 7.5), and disrupted by sonic oscillation. The suspension of disrupted cells was centrifuged at 20,000 g for 30 minutes to remove cellular debris. The crude extracts thus obtained were stored at --15°C. Nucleic Acid Precipitation. The protein concentration of the crude extract was adjusted to approximately 15 mg/ml with 0.05 M potassium phosphate buffer (pH 7.5), and solid ammonium sulfate was added to a final concentration of 0.20 M. The nucleic acids were then precipitated by the dropwide addition of 0.20 volume of 1.0% protamine. The enzyme is easily precipitated with protamine and, hence, this step must be run carefully. First Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to the supernatant from the protamine step to 50% saturation, centrifuged, and the precipitate discarded. The supernatant was adjusted to 65% saturation with solid ammonium sulfate, centrifuged, and the precipitate dissolved and adjusted to twice the original volume with water. Fractionation with Calcium Phosphate Gel. Calcium phosphate gel was added in a stepwise fashion until about 80% of the enzyme was adsorbed. The gel was then washed with 0.07 M phosphate buffer, pH 7.5, and then the enzyme was eluted from the gel by washing twice with 3C. A. Hirsch, M. Rasminsky, B. D. Davis, and E. C. C. Lin, J. Biol. Chem. 3770 (1963). ' R. J. Pennington, Biochem. J. 80, 649 (1961). 5Obtained from Dr. Marvin Bryant, Department of Dairy Science, University of Illinois, Urbana, Illinois. eD. J'. Walker, Biochem. J. 69, 524 (1958).
[122]
LACTYL-COA DEHYDRASE
685
volumes of 0.12M phosphate buffer, pH 7.5, equal to about twice the volume of the gel. Second Ammonium Sul]ate Fractionation. The eluates from the calcium phosphate gel were combined and solid ammonium sulfate added to a final concentration of 55% saturation. The precipitate was discarded and the ammonium sulfate concentration adjusted to 70% saturation. The precipitate was collected and resuspended in 0.5 M phosphate buffer, pH 7.5. A summary of the procedure is shown in the accompanying table. PURIFICATION PROCEDURE
Fraction Crude extract Protamine First ammonium sulfate Calcium phosphate gel Second ammonium sulfate
Total Volume activity (ml) (units) 15 35 60 17 10
2.76 2.58 2.20 1.50 1.10
Specific activity (units/mg Yield Protein protein (%) (mg) X 10t) 93 80 54 40
480 420 126 37 16
O. 58 O. 61 1.75 4.05 6.90
Properties The enzyme has not been purified sufficiently to study in detail the kinetics of the reaction and the effects of inhibitors and activators. The pH optimum for the reaction appears to be between 7.5 and 8.0. The activity observed with crotonyl-CoA as substrate is 75% that observed with acrylyl-CoA. EDTA does not inhibit, and sometimes stimulates, activity. The enzyme is relatively stable in crude extracts stored at --15 ° . However, it becomes very unstable during purification. For example, approximately 10% of the enzyme activity is lost per hour when the enzyme is allowed to stand at 0 ° after the first ammonium sulfate step.