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Sherry L. Mowbray – One of the best experts on this subject based on the ideXlab platform.

  • d ribose 5 phosphate isomerase b from escherichia coli is also a functional d allose 6 phosphate isomerase while the mycobacterium tuberculosis enzyme is not
    Journal of Molecular Biology, 2008
    Co-Authors: Annette K. Roos, S Mariano, Eva Kowalinski, Laurent Salmon, Sherry L. Mowbray
    Abstract:

    Interconversion of D-ribose-5-phosphate (R5P) and D-ribulose-5-phosphate is an important step in the pentose phosphosphatehway. Two unrelated Enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-phosphate (All6P) and D-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme’s primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme’s structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.

Annette K. Roos – One of the best experts on this subject based on the ideXlab platform.

  • d ribose 5 phosphate isomerase b from escherichia coli is also a functional d allose 6 phosphate isomerase while the mycobacterium tuberculosis enzyme is not
    Journal of Molecular Biology, 2008
    Co-Authors: Annette K. Roos, S Mariano, Eva Kowalinski, Laurent Salmon, Sherry L. Mowbray
    Abstract:

    Interconversion of D-ribose-5-phosphate (R5P) and D-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated Enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-phosphate (All6P) and D-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme’s primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme’s structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.

Vernon E. Anderson – One of the best experts on this subject based on the ideXlab platform.

  • Multiple alternative substrate kinetics Proteins and proteomics
    Biochimica et Biophysica Acta, 2020
    Co-Authors: Vernon E. Anderson
    Abstract:

    The specificity of Enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme’s specificity using kinetics have been utilized over the decades. Fersht’s definition of the ratio kcat/Km for two different substrates as the “specificity constant” (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for Enzymes obeying Michaelis–Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment.

  • Multiple alternative substrate kinetics
    Biochimica et Biophysica Acta – Proteins and Proteomics, 2015
    Co-Authors: Vernon E. Anderson
    Abstract:

    The specificity of Enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme’s specificity using kinetics have been utilized over the decades. Fersht’s definition of the ratio kcat/Km for two different substrates as the “specificity constant” (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for Enzymes obeying Michaelis-Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment.

Philip W. Kuchel – One of the best experts on this subject based on the ideXlab platform.

  • review of mutarotase in metabolic subculture and analytical biochemistry prelude to 19f nmr studies of its substrate specificity and mechanism
    Australian Journal of Chemistry, 2020
    Co-Authors: Dmitry Shishmarev, Lucas Quiquempoix, Clement Q Fontenelle, Bruno Linclau, Philip W. Kuchel
    Abstract:

    This is the first paper in a sequential pair devoted to the enzyme mutarotase (aldose 1-epimerase; EC 5.1.3.3). Here, the broader context of the physiological role of mutarotase, among those Enzymes considered to be part of ‘metabolic structure’, is reviewed. We also summarise the current knowledge about the molecular mechanism and substrate specificity of the enzyme, which is considered in the context of the binding of fluorinated glucose analogues to the enzyme’s active site. This was done as a prelude to our experimental studies of the anomerisation of fluorinated sugars by mutarotase that are described in the following paper.

  • Review of Mutarotase in ‘Metabolic Subculture’ and Analytical Biochemistry: Prelude to 19F NMR Studies of its Substrate Specificity and Mechanism
    Australian Journal of Chemistry, 2020
    Co-Authors: Dmitry Shishmarev, Lucas Quiquempoix, Clement Q Fontenelle, Bruno Linclau, Philip W. Kuchel
    Abstract:

    This is the first paper in a sequential pair devoted to the enzyme mutarotase (aldose 1-epimerase; EC 5.1.3.3). Here, the broader context of the physiological role of mutarotase, among those Enzymes considered to be part of ‘metabolic structure’, is reviewed. We also summarise the current knowledge about the molecular mechanism and substrate specificity of the enzyme, which is considered in the context of the binding of fluorinated glucose analogues to the enzyme’s active site. This was done as a prelude to our experimental studies of the anomerisation of fluorinated sugars by mutarotase that are described in the following paper.

Eva Kowalinski – One of the best experts on this subject based on the ideXlab platform.

  • d ribose 5 phosphate isomerase b from escherichia coli is also a functional d allose 6 phosphate isomerase while the mycobacterium tuberculosis enzyme is not
    Journal of Molecular Biology, 2008
    Co-Authors: Annette K. Roos, S Mariano, Eva Kowalinski, Laurent Salmon, Sherry L. Mowbray
    Abstract:

    Interconversion of D-ribose-5-phosphate (R5P) and D-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated Enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-phosphate (All6P) and D-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme’s primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme’s structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.