The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-aspa... more The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been measured over the range pH 4.5 to pH 9.1 by a direct spectrophotometric assay at 220 nm and by a coupled assay utilizing glutamate dehydrogenase to detect the ammonia produced. The velocity of the hydrolysis reaction at saturating levels of substrate is independent of pH over this interval. The plot of V/km over the same interval is bell-shaped, being dependent on pKa values of 6.58 and 8.69. The higher pKa is attributed to the amino group of asparagine. The lower pKa is associated with the enzyme active site and is probably due to an imidazole group.
In a previous study (Melzer and O'Leary, 1987, Plant Physiol. 84, 58-60), we used isotopi... more In a previous study (Melzer and O'Leary, 1987, Plant Physiol. 84, 58-60), we used isotopic methods to show that a substantial fraction of protein-bound aspartic acid in tobacco is derived from anaplerotic synthesis via phosphoenolpyruvate (PEP) carboxylase. Similar studies in soybean (Glycine max L.) and spinach (Spinacia oleracea L.) showed a similar pattern, and this pattern persists with age because of slow protein turnover. A more quantitative analysis indicates that about 40% of protein-bound aspartate is derived in this manner. Analyses of free aspartic and malic acids show that contribution of PEP carboxylase to the synthesis of these acids decreases with increasing age. The C4 plant Zea mays L. did not show this pattern.
Carbonyl oxygen kinetic isotope effects were measured on the alkaline hydrolysis and hydrazinolys... more Carbonyl oxygen kinetic isotope effects were measured on the alkaline hydrolysis and hydrazinolysis of methyl benzoate. (methyl-/sup 13/C) Methyl (carbonyl-/sup 18/O)-benzoate, 90 percent enriched at both positions, was mixed with natural abundance methyl benzoate in the ratio of 1 : 4. This substrate was subjected to hydrolysis or hydrazinolysis and the /sup 13/C//sup 12/C isotope ratio of the product methanol was measured at 30 percent reaction and at 100 percent reaction. Because of the nature of the double-labeling method, the apparent carbon isotope effect obtained by this measurement is actually the product of the methyl carbon isotope effect and the carbonyl isotope effect. The former isotope effect is nearly unity; so the measurements provide the carbonyl oxygen isotope effect directly. Data are summarized. Small corrections have been included. 13 references.
A double-labeling procedure has been devised for use in measurement of heavy-atom isotope effects... more A double-labeling procedure has been devised for use in measurement of heavy-atom isotope effects. This method is of particular value for measurement of isotope effects at sites which are not easily amenable to study by standard isotope-ratio techniques. Substrate is synthesized which is highly labeled at two positions - one the position of interest in the isotope effect experiment and the other a position whose isotope effect is easily measured by standard isotope-ratio methods. This labeled substrate is mixed with unlabeled substrate, and the isotope effect is measured for the measurable site. This apparent isotope effect is actually the product of the isotope effect at the measurable site and that at the site of interest. Separate measurement of the former isotope effect then permits calculation of the isotope effect at the site of interest. Heavy-atom isotope effects on the hydrolysis and hydrazinolysis of methyl benzoate at 25/sup 0/C in aqueous solution have been measured, using the methyl carbon atom. In the alkaline hydrolysis the carbonyl oxygen isotope effect is k/sup 16//k/sup 18/ = 1.0046 +- 0.0020; the carbonyl carbon isotope effect is k/sup 12/k/sup 13/ = 1.0426 +- 0.0026; the ether oxygen isotope effect is k/sup 16//k/sup 18/ = 1.0062more » +- 0.0006; the methyl carbon isotope effect is k/sup 12//k/sup 13/ = 1.0004 +- 0.0005. For the hydrozinolysis at pH 7.9 the valuesare carbonyl oxygen, 1.0184 +- 0.0014; carbonyl carbon, 1.0410 +- 0.0022; ether oxygen, 1.0413 +- 0.0028; methyl carbon 1.0022 +- 0.0004. These isotope effects indicate that the rate-determining step in the alkaline hydrolysis is the formation of the tetrahedral intermediate. The small magnitudes of the oxygen isotope effects require that the transition state in this step be relatively reactant-like. The isotope effects on the hydrazinolysis indicate that the decomposition of the tetrahedral intermediate is rate determining and that the transition state for this step is relatively product-like.« less
... (20) ML Bender, GE Clement, FJ Kezdy, and H. d'A. Heck, (21) ML Bender, MJ Gibian. and D... more ... (20) ML Bender, GE Clement, FJ Kezdy, and H. d'A. Heck, (21) ML Bender, MJ Gibian. and DJWhelan, Proc. Nat. ibid., 86, 3680 (1964). Acad. Sci., U. S., 56, 833 (1966). ... Org. Chem., 5,237 (1967). (36) ML Bender, RD Ginger, and JP Unik, J. Amer. Chem. Soc., 80, 1044 (1958). ...
... (11) Nguyen, MT; Ha ... In an earlier study, we reported H/D exchange in systems in which the... more ... (11) Nguyen, MT; Ha ... In an earlier study, we reported H/D exchange in systems in which the reactant ion contains only one exchangeable proton or deu-teron, reactions such as DO-with NH3 and HO-with C6D6.' Similar studies on H/D scrambling in positive ion reactions have ...
The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-aspa... more The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been measured over the range pH 4.5 to pH 9.1 by a direct spectrophotometric assay at 220 nm and by a coupled assay utilizing glutamate dehydrogenase to detect the ammonia produced. The velocity of the hydrolysis reaction at saturating levels of substrate is independent of pH over this interval. The plot of V/km over the same interval is bell-shaped, being dependent on pKa values of 6.58 and 8.69. The higher pKa is attributed to the amino group of asparagine. The lower pKa is associated with the enzyme active site and is probably due to an imidazole group.
In a previous study (Melzer and O'Leary, 1987, Plant Physiol. 84, 58-60), we used isotopi... more In a previous study (Melzer and O'Leary, 1987, Plant Physiol. 84, 58-60), we used isotopic methods to show that a substantial fraction of protein-bound aspartic acid in tobacco is derived from anaplerotic synthesis via phosphoenolpyruvate (PEP) carboxylase. Similar studies in soybean (Glycine max L.) and spinach (Spinacia oleracea L.) showed a similar pattern, and this pattern persists with age because of slow protein turnover. A more quantitative analysis indicates that about 40% of protein-bound aspartate is derived in this manner. Analyses of free aspartic and malic acids show that contribution of PEP carboxylase to the synthesis of these acids decreases with increasing age. The C4 plant Zea mays L. did not show this pattern.
Carbonyl oxygen kinetic isotope effects were measured on the alkaline hydrolysis and hydrazinolys... more Carbonyl oxygen kinetic isotope effects were measured on the alkaline hydrolysis and hydrazinolysis of methyl benzoate. (methyl-/sup 13/C) Methyl (carbonyl-/sup 18/O)-benzoate, 90 percent enriched at both positions, was mixed with natural abundance methyl benzoate in the ratio of 1 : 4. This substrate was subjected to hydrolysis or hydrazinolysis and the /sup 13/C//sup 12/C isotope ratio of the product methanol was measured at 30 percent reaction and at 100 percent reaction. Because of the nature of the double-labeling method, the apparent carbon isotope effect obtained by this measurement is actually the product of the methyl carbon isotope effect and the carbonyl isotope effect. The former isotope effect is nearly unity; so the measurements provide the carbonyl oxygen isotope effect directly. Data are summarized. Small corrections have been included. 13 references.
A double-labeling procedure has been devised for use in measurement of heavy-atom isotope effects... more A double-labeling procedure has been devised for use in measurement of heavy-atom isotope effects. This method is of particular value for measurement of isotope effects at sites which are not easily amenable to study by standard isotope-ratio techniques. Substrate is synthesized which is highly labeled at two positions - one the position of interest in the isotope effect experiment and the other a position whose isotope effect is easily measured by standard isotope-ratio methods. This labeled substrate is mixed with unlabeled substrate, and the isotope effect is measured for the measurable site. This apparent isotope effect is actually the product of the isotope effect at the measurable site and that at the site of interest. Separate measurement of the former isotope effect then permits calculation of the isotope effect at the site of interest. Heavy-atom isotope effects on the hydrolysis and hydrazinolysis of methyl benzoate at 25/sup 0/C in aqueous solution have been measured, using the methyl carbon atom. In the alkaline hydrolysis the carbonyl oxygen isotope effect is k/sup 16//k/sup 18/ = 1.0046 +- 0.0020; the carbonyl carbon isotope effect is k/sup 12/k/sup 13/ = 1.0426 +- 0.0026; the ether oxygen isotope effect is k/sup 16//k/sup 18/ = 1.0062more » +- 0.0006; the methyl carbon isotope effect is k/sup 12//k/sup 13/ = 1.0004 +- 0.0005. For the hydrozinolysis at pH 7.9 the valuesare carbonyl oxygen, 1.0184 +- 0.0014; carbonyl carbon, 1.0410 +- 0.0022; ether oxygen, 1.0413 +- 0.0028; methyl carbon 1.0022 +- 0.0004. These isotope effects indicate that the rate-determining step in the alkaline hydrolysis is the formation of the tetrahedral intermediate. The small magnitudes of the oxygen isotope effects require that the transition state in this step be relatively reactant-like. The isotope effects on the hydrazinolysis indicate that the decomposition of the tetrahedral intermediate is rate determining and that the transition state for this step is relatively product-like.« less
... (20) ML Bender, GE Clement, FJ Kezdy, and H. d'A. Heck, (21) ML Bender, MJ Gibian. and D... more ... (20) ML Bender, GE Clement, FJ Kezdy, and H. d'A. Heck, (21) ML Bender, MJ Gibian. and DJWhelan, Proc. Nat. ibid., 86, 3680 (1964). Acad. Sci., U. S., 56, 833 (1966). ... Org. Chem., 5,237 (1967). (36) ML Bender, RD Ginger, and JP Unik, J. Amer. Chem. Soc., 80, 1044 (1958). ...
... (11) Nguyen, MT; Ha ... In an earlier study, we reported H/D exchange in systems in which the... more ... (11) Nguyen, MT; Ha ... In an earlier study, we reported H/D exchange in systems in which the reactant ion contains only one exchangeable proton or deu-teron, reactions such as DO-with NH3 and HO-with C6D6.' Similar studies on H/D scrambling in positive ion reactions have ...
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