The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site‐specific mutagenesis

Holtz, Kathleen M.; Kantrowitz, Evan R.

doi:10.1016/s0014-5793(99)01448-9

Cited by 145 publications

(125 citation statements)

References 46 publications

(55 reference statements)

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“…The mixture was incubated at 33°C with shaking by a rotary shaker (100 rev/min) and after that was centrifuged for 20 min at 10000 g and 0-4C to remove the cells. 7.0. The suspension was shaken at 5°C overnight and then centrifuged at 60 000 g for 2 h.…”

Section: Culture Methodsmentioning

confidence: 99%

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Purification and Properties of Alkaline Phosphatase fromBacillus Cereus

Kostadinova

Marhova

2010

Biotechnology & Biotechnological Equipment

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Section: Culture Methodsmentioning

confidence: 99%

“…Alkaline phosphatases produced by B.subtilis and E.coli, respectively were found to be metal proteins and needed metal ions for enzyme expression (7,10). Our results compared B. cereus APs with enzymes of other Bacillus.…”

Section: Effect Of Metal Ions and Edtamentioning

confidence: 99%

Purification and Properties of Alkaline Phosphatase fromBacillus Cereus

Kostadinova

Marhova

2010

Biotechnology & Biotechnological Equipment

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“…The work on the reaction mechanism of AP is mostly based on structural, kinetic, and mutational studies on the enzyme from E. coli [458,595]. In contrast to many other metallophosphatases, which activate the water molecule for a direct attack on the substrate, the reaction mechanism of AP is special in that it proceeds via a covalent intermediate [596] (Fig.…”

Section: Catalytic Mechanismmentioning

confidence: 99%

Cellular function and molecular structure of ecto-nucleotidases

Zimmermann

Zebisch

Sträter

2012

Purinergic Signalling

872

922

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Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ectonucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5′-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

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“…As a class, the students are shown and guided through a published figure that illustrates the general mechanism of alkaline phosphatase [6]. To facilitate student learning, the published mechanism figure (Fig.…”

Section: Part Ii: Correlation Of Kinetic Data and Amino Acid Functionmentioning

confidence: 99%

“…In the last part of the exercise, students use information gained from the structure of alkaline phosphatase's active site, the general mechanism, and published kinetic data for various active site amino acid mutations (Table I) to propose specific roles for certain amino acids [6][7][8][9]. They are asked to answer the following questions for Ser102, Asp327, and His412.…”

Section: Part Ii: Correlation Of Kinetic Data and Amino Acid Functionmentioning

confidence: 99%

Improvement of student understanding of how kinetic data facilitates the determination of amino acid catalytic function through an alkaline phosphatase structure/mechanism bioinformatics exercise

Grunwald

Krueger

2008

Biochem Molecular Bio Educ

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Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was developed and implemented. In this exercise, students examine the structure of alkaline phosphatase using the free, on-line bioinformatics protein-modeling program Protein Explorer. Specifically, students examine the active site residues of alkaline phosphatase and propose functions for these residues. Furthermore, by examining the mechanism of alkaline phosphatase and by using the published kinetic data, students propose specific roles for several active-site residues. Paired t-test analysis of pre-versus postexercise assessment data shows that the completion of the exercise improves student's ability to use kinetic data correctly thereby determining a probable catalytic function for an active site amino acid.Keywords: Alkaline phosphatase, bioinformatics, assessment, enzyme kinetics.The American Society for Biochemistry and Molecular Biology (ASBMB) Recommended Biochemistry and Molecular Biology Undergraduate Curriculum lists kinetics as a core-content item that biochemistry and molecular biology students should master before receiving their baccalaureate degree [1]. Enzyme kinetics has traditionally been a major focus in both the lecture and laboratory curriculum for biochemistry courses. Laboratory exercises that utilize alkaline phosphatase as a model enzyme to illustrate steady-state kinetic properties of enzymes, including determining K m and V max , and the inhibition constants for various substances have been developed and used extensively in the undergraduate biochemistry laboratory [2,3]. Alkaline phosphatase's stability, straightforward assay, and inexpensive assay reagents make it an ideal enzyme for teaching enzyme kinetics in the biochemistry laboratory. This traditional laboratory exercise not only helps students learn how to experimentally determine enzyme kinetic constants but also reinforces the enzyme kinetics material that is covered extensively in the lecture portion of a biochemistry course.At the University of Wisconsin-La Crosse (UW-L), the one-semester Survey of Biochemistry course includes seven 55-minute lecture periods devoted to enzymes in which the following topics are covered: 1) general properties of enzymes, 2) effect of enzymes on activation energy and reaction coordinates, 3) catalytic mechanisms, specifically focusing on chymotrypsin, and 4) enzyme kinetics. The enzyme-kinetics section includes four lectures covering chemical kinetics, derivation of the Michaelis-Menten equation, analysis of kinetic data, and enzyme inhibition. The course also includes one 3-hour laboratory section in which the students experimentally determine the K m and V max values for the interaction of the enzyme, alkaline phosphatase, and the substrate, pnitroph...

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The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site‐specific mutagenesis

Cited by 145 publications

References 46 publications

Purification and Properties of Alkaline Phosphatase fromBacillus Cereus

Purification and Properties of Alkaline Phosphatase fromBacillus Cereus

Cellular function and molecular structure of ecto-nucleotidases

Improvement of student understanding of how kinetic data facilitates the determination of amino acid catalytic function through an alkaline phosphatase structure/mechanism bioinformatics exercise

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