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. 1969 Sep;114(2):243–251. doi: 10.1042/bj1140243

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

S E Halford 1, N G Bennett 1, D R Trentham 1, H Gutfreund 1
PMCID: PMC1184849  PMID: 4897458

Abstract

1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme–phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22° the rate constant is 167sec.−1 for the rearrangement of the initially formed binary complex and is 18sec.−1 for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme–substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme–phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ECHOLS H., GAREN A., GAREN S., TORRIANI A. Genetic control of repression of alkaline phosphatase in E. coli. J Mol Biol. 1961 Aug;3:425–438. doi: 10.1016/s0022-2836(61)80055-7. [DOI] [PubMed] [Google Scholar]
  2. Fernley H. N., Walker P. C. Effect of sodium chloride on Escherichia coli alkaline phosphatase. Biochem J. 1968 Nov;110(2):11P–12P. doi: 10.1042/bj1100011pb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fernley H. N., Walker P. G. Studies on alkaline phosphatase. Transient-state and steady-state kinetics of Escherichia coli alkaline phosphatase. Biochem J. 1969 Jan;111(2):187–194. doi: 10.1042/bj1110187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. KOCHMAN M., MASTALERZ P., WOLNA E. PHOSPHONIC ACIDS--A NEW GROUP OF COMPETITIVE INHIBITORS OF INTESTINAL ALKALINE PHOSPHATASE. Arch Immunol Ther Exp (Warsz) 1964;12:106–112. [PubMed] [Google Scholar]
  5. Ko S. H., Kézdy F. J. The kinetics of the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 2,4-dinitrophenyl phosphate. J Am Chem Soc. 1967 Dec 20;89(26):7139–7140. doi: 10.1021/ja01002a068. [DOI] [PubMed] [Google Scholar]
  6. MALAMY M. H., HORECKER B. L. PURIFICATION AND CRYSTALLIZATION OF THE ALKALINE PHOSPHATASE OF ESCHERICHIA COLI. Biochemistry. 1964 Dec;3:1893–1897. doi: 10.1021/bi00900a018. [DOI] [PubMed] [Google Scholar]
  7. MALAMY M. H., HORECKER B. L. RELEASE OF ALKALINE PHOSPHATASE FROM CELLS OF ESCHERICHIA COLI UPON LYSOZYME SPHEROPLAST FORMATION. Biochemistry. 1964 Dec;3:1889–1893. doi: 10.1021/bi00900a017. [DOI] [PubMed] [Google Scholar]
  8. PLOCKE D. J., VALLEE B. L. Interaction of alkaline phosphatase of E. coli with metal ions and chelating agents. Biochemistry. 1962 Nov;1:1039–1043. doi: 10.1021/bi00912a014. [DOI] [PubMed] [Google Scholar]
  9. Reynolds J. A., Schlesinger M. J. Conformational states of the subunit of Escherichia coli alkaline phosphatase. Biochemistry. 1967 Nov;6(11):3552–3559. doi: 10.1021/bi00863a029. [DOI] [PubMed] [Google Scholar]
  10. STOCKELL A. The binding of diphosphopyridine nucleotide by yeast glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem. 1959 May;234(5):1286–1292. [PubMed] [Google Scholar]
  11. Schlesinger M. J., Andersen L. Multiple molecular forms of the alkaline phosphatase of Escherichia coli. Ann N Y Acad Sci. 1968 Jun 14;151(1):159–170. doi: 10.1111/j.1749-6632.1968.tb11886.x. [DOI] [PubMed] [Google Scholar]
  12. Schlesinger M. J., Barrett K. The reversible dissociation of the alkaline phosphatase of Escherichia coli. I. Formation and reactivation of subunits. J Biol Chem. 1965 Nov;240(11):4284–4292. [PubMed] [Google Scholar]
  13. Simpson R. T., Vallee B. L. Two differentiable classes of metal atoms in alkaline phosphatase of Escherichia coli. Biochemistry. 1968 Dec;7(12):4343–4350. doi: 10.1021/bi00852a029. [DOI] [PubMed] [Google Scholar]
  14. Trentham D. R., Gutfreund H. The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli. Biochem J. 1968 Jan;106(2):455–460. doi: 10.1042/bj1060455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Wenn R. V., Williams J. The isoelectric fractionation of hen's-egg ovotransferrin. Biochem J. 1968 Jun;108(1):69–74. doi: 10.1042/bj1080069. [DOI] [PMC free article] [PubMed] [Google Scholar]

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