Kinetics and Inhibition of Hydrolysis of Para-Nitrophenyl Phosphate by Wheat Germ Acid Phosphatase

Pages: 7 (2221 words) Published: February 19, 2014
Kinetics and Inhibition of Hydrolysis of Para-Nitrophenylphosphate by Wheat Germ Acid Phosphatase Ryan Austin, and Ali Hussain
Department of Chemistry, Bloomsburg University, Bloomsburg, PA 17815

ABSTRACT: Qualitative methods were used to the enzyme catalyzed hydrolysis of para-nitrophenylphosphate (p-NPP) to para-nitrophenol (p-NP) by wheat germ acid phosphatase (WGAP). The optimum pH was determined by examining the amount of p-NP formed due to enzymatic activity of solutions of 2.5 mM p-NPP varied over a range of pH from pH 3 to 8; the optimum pH was 4.5. Additional methods examined WGAP activity under uninhibited and inhibited conditions with the addition of 10.0 mM molybdate ion. Michaelis-Menten plots for both sets of data and Lineweaver-Burk plots were examined to determine the type of inhibition and to compare the accuracy of both. By Michaelis-Menten, the Km (M) was 0.0004 ± 4.9800 * 10-5 M (uninhibited) and 0.0038 ± 0.0011 M (inhibited). The Vmax were 0.0771 ± 0.0018 μmol min-1 (uninhibited) and 0.0100 ± 0.0012 μmol min-1 (inhibited) for the reactions. The turnover numbers were 38.6 min-1 and 5.00 min-1 for the uninhibited and inhibited reactions. By Lineweaver-Burk, the Km were 0.00035 M and 0.0062 M for the uninhibited and inhibited reactions, respectively. Also, the Vmax were 0.10 μmol * min-1 and 0.0054 μmol * min-1 for the uninhibited and the inhibited reactions. The kcat were 37.5 min-1 and 2.7 min-1 for the uninhibited and inhibited reactions. From these data the kcat/Km were 96,500 M-1min-1 and 1,320 M-1min-1 for the uninhibited and inhibited reactions by Michaelis-Menten and 106,383 M-1min-1 and 438.1 M-1min-1 for the uninhibited and inhibited reactions by Lineweaver-Burk. By Michaelis-Menten and Lineweaver-Burk, the uninhibited reaction data showed a lower Km indicating greater affinity of the substrate for wheat germ acid phosphatase. Both Vmax and kcat were higher indicating faster production of the product and a higher turnover number indicating a higher maximum number of substrate molecules being turned into product per minute. The higher kcat/Km for the uninhibited reaction indicated that WGAP was more catalytically efficient and was characterized with a higher turnover of substrate molecules into product per concentration of enzyme. The kinetics values indicated that the molybdate ion acted as a mixed inhibitor due to the decreased Vmax and increased Km. The calculated inhibition constant, Ki was 0.0103 M and indicated the inhibitor preferred binding to WGAP.

EXPERIMENTAL PROCEDURES
The procedure published by Pugh, et al. (1) was followed. Deviations from standard procedure are summarized as follows: Buffer pH. When determining the kinetic constants for hydroloysis of p-NPP without inhibitor, the citrate buffer used had a pH of 4.5 instead of a pH of 4.8.

Determination of kinetic constants for hydrolysis of p-NPP with inhibitor. The inhibitor used was 10.0 mM molybdate ion.

RESULTS AND DISCUSSION

To study the enzyme catalyzed reaction of hydrolysis of p-NPP by WGAP, a quantitative analysis of enzyme kinetics was utilized. Reaction rates, mechanisms, and the specificity for para-nitrophenylphosphate along with the addition of molybdate as an inhibitor were observed by reading A405. Through Michaelis-Menten kinetics, WGAP behavior was studied through plotting reaction velocity, versus. By generating a Lineweaver-Burk plot, Vmax and Km were calculated for the uninhibited reaction and the molybdate ion inhibited reaction.

The standard curves. The A405 of p-NP at was analyzed over a range of concentrations to produce standard curves, as shown in Figure 1. The best fit lines of the standard curves were both linear (Figure 1). The absorbance values of the experimental data fit on a linear portion of the standard curve. A405 plotted against µmol p-NP had an R2 value of 0.9989 during the determination of the optimum pH. The standard curve for the hydrolysis of...

References: 1. Pugh, M. E., Schultz, E., Bell, T. T., and Borland M. G. (2012) Kinetics and inhibition of hydrolysis of para-nitrophenylphosphate by wheat germ acid phosphatase, in Concepts and Techniques in the Biochemistry 1 Laboratory (Pugh, M. E., Schultz, E., Bell, T. T., and Borland M. G., eds.) Fall 2013 ed., pp 104-114, Bloomsburg University Press, Bloomsburg PA.
2. Joyce, B. K., & Grisolia, S. (1960). Purification and properties of a nonspecific acid phosphatase from wheat germ. J. biol. Chem, 235(2278), 1960.
3. McKee, T., and McKee, J. R. (2012) Enzymes, in Biochemistry: The Molecular Basis of Life, Fifth ed., pp 192-199, Oxford University Press, New York.
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