Browsing by Author "CARDEMIL, E"

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    CHEMICAL MODIFICATION OF ARGINYL RESIDUES IN RABBIT MUSCLE PYRUVATE-KINASE
    (1981) CARDEMIL, E; EYZAGUIRRE, J
    Rabbit muscle pyruvate kinase was inactivated with the arginine-specific reagent 2,3-butanedione, in borate buffer, following psuedo-first-order kinetics. Graphical representation of the kobs as a function of inactivator concentration follows a straight line which is compatible with the following mechanism: .**GRAPHIC**. Values for k1 = 4.6 M-1 min-1 and k1 = 0.0022 min-1 can be estimated. The order of the reaction was near 1, suggesting that the modification of a single amino acid residue of the enzyme is responsible for its inactivation. Almost full catalytic activity was recovered when excess butanedione and borate buffer were removed by Sephadex G-25 gel filtration, which suggests that only arginyl residues were modified. The inactivation was partially prevented by phosphoenolypyruvate in the presence of K+ and Mg2+, but not by the competitive inhibitors lactate and bicarbonate, or ADP, with or without added metal ions. Mg2+ alone increased the rate of inactivation. Quantification of residual arginine residues after chemical modification, using amino acid analysis, gives no precise correlation between the number of residues modified and the residual activity; however, .apprx. 4 residues react per enzyme subunit, showing the presence of reactive, nonessential arginines. One essential arginyl residue is present per subunit in rabbit muscle pyruvate kinase, and this residue is probably located near the binding site of the phosphate group of phosphoenolpyruvate.
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    CHEMICAL MODIFICATION OF ESSENTIAL ARGININES IN PIG-LIVER PHOSPHOMEVALONATE KINASE
    (1982) VERGARA, M; ALVEAR, M; CARDEMIL, E; JABALQUINTO, AM; EYZAGUIRRE, J
    Phosphomevalonate [MVAP] kinase, an enzyme of the polyisoprenoid biosynthesis pathway, catalyzes the transfer of phosphate from ATP to MVAP, with the formation of pyrophosphomevalonate and ADP. The pig liver enzyme, a monomer of MW 22,000, possesses 1 cysteinyl residue, which is essential for catalysis. By means of chemical modification of a partially purified preparation, the participation of arginine residues in the enzyme active site was studied. Butanedione and phenylglyoxal were chosen as group-specific reagents. The kinetics of inactivation by both reagents is rather complex, suggesting that several arginine residues, directly or indirectly related to the active site, are being modified. Both substrates, MVAP and Mg-ATP, protect against inactivation but to a different extent, depending on the modifier used. With butanedione, almost total protection is achieved with Mg-ATP. Better protection with MVAP is observed for the modification with phenylglyoxal, but with this reagent, Mg-ATP protects strongly only at very high concentrations. A reversible inactivation mechanism is followed with butanedione, while for phenylglyoxal this mechanism appears to be irreversible, in agreement with findings by other authors. MVAP kinase probably presents 2 or more arginyl residues in or near its active site, one of them being involved in the binding of Mg-ATP, and at least another located in the neighborhood of the MVAP binding site. A more precise determination of the number of essential residues requires its measurement by chemical methods, utilizing a homogeneous enzyme preparation.
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    EVIDENCE OF ESSENTIAL ARGINYL RESIDUES IN RABBIT MUSCLE PYRUVATE-KINASE
    (1979) CARDEMIL, E; EYZAGUIRRE, J
    Rabbit muscle pyruvate kinase [EC 2.7.1.40] is inactivated by 2,3-butanedione in borate buffer. The inactivation follows pseudo-1st-order kinetics with a calculated 2nd-order rate constant of 4.6/M per min. The modification can be reversed with almost total recovery of activity by elimination of the butanedione and borate buffer, suggesting that only arginyl groups are modified. This result agrees with the loss of arginine detected by amino acid analysis of the modified enzyme. Using the kinetic data, it was estimated that the reaction of a single butanedione molecule/subunit of the enzyme is enough to completely inactivate the protein. The inactivation is partially prevented by phosphoenolpyruvate in the presence of K+ and Mg2+, but not by the competitive inhibitors lactate and bicarbonate. These findings point to an essential arginyl residue being located near the phosphate binding site of phosphoenolpyruvate.
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    PURIFICATION AND CHARACTERIZATION OF AVIAN LIVER MEVALONATE-5-PYROPHOSPHATE DECARBOXYLASE
    (1982) ALVEAR, M; JABALQUINTO, AM; EYZAGUIRRE, J; CARDEMIL, E
    Mevalonate-5-pyrophosphate decarboxylase was purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of MW 85,400 .+-. 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0-6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column choromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM were obtained for mevalonate-5-pyrophosphate and ATP, respectively.