The Statistical Analysis of Enzyme Kinetic Data

Cleland, W. W.

doi:10.1002/9780470122747.ch1

Cited by 716 publications

(359 citation statements)

References 12 publications

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“…Velocities were determined in duplicate at each of the 16 conditions defined by a 4 ϫ 4 matrix of acceptor and nucleotide concentrations. The averaged data were fit using the SEQUENO program, which uses a weighted least square fitting algorithm (25).…”

Section: Methodsmentioning

confidence: 99%

Testing the Sulfotransferase Molecular Pore Hypothesis

Cook

Wang

Almo

et al. 2013

Journal of Biological Chemistry

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Background: Human cytosolic sulfotransferases (SULTs) regulate the bioactivities of hundreds of signaling molecules. Results: Mutants define the structural changes that determine substrate selectivity. Conclusion: Substrates enter the active site through a molecular pore that opens and closes in response to nucleotide binding. Significance: SULT selectivity is a fundamental determinant of sulfur biology.

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Section: Methodsmentioning

confidence: 99%

Testing the Sulfotransferase Molecular Pore Hypothesis

Cook

Wang

Almo

et al. 2013

Journal of Biological Chemistry

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“…To compare competitive, noncompetitive, and uncompetitive models statistically, each pair was contrasted by using Akaike's method (14) by using GraphPad Prism, which reports the relative percent chance that a model is correct. To determine best-fit K is and K ii values for a mixed inhibition model, data were globally fitted to a mixed inhibition model with the program KinetAsyst (Intellikinetics), which uses the algorithm of Cleland (15).…”

Section: Methodsmentioning

confidence: 99%

Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

Lü

Bernstein²,

Satyshur³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.

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“…Initial velocities for each reaction were determined by plotting the SAH concentration versus time and were fit to the Michaelis-Menten equation v ¼ VA/(K þ A). The kinetic constants were calculated using non-linear regression, 15 where K was the Michaelis constant for the varied substrate and V was the maximum velocity.…”

Section: Activity Assaysmentioning

confidence: 99%

Probing the catalytic mechanism of a C‐3′‐methyltransferase involved in the biosynthesis of D‐tetronitrose

Bruender

Holden

2012

Protein Science

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D-Tetronitrose is a nitro-containing tetradeoxysugar found attached to the antitumor and antibacterial agent tetrocarcin A. The biosynthesis of this highly unusual sugar in Micromonospora chalcea requires 10 enzymes. The fifth step in the pathway involves the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the C-3 0 carbon of dTDP-3-amino-2,3,6-trideoxy-4-keto-Dglucose. The enzyme responsible for this transformation is referred to as TcaB9. It is a monomeric enzyme with a molecular architecture based around three domains. The N-terminal motif contains a binding site for a structural zinc ion. The middle-and C-terminal domains serve to anchor the SAM and dTDP-sugar ligands, respectively, to the protein, and the active site of TcaB9 is wedged between these two regions. For this investigation, the roles of Tyr 76, His 181, Tyr 222, Glu 224, and His 225, which form the active site of TcaB9, were probed by site-directed mutagenesis, kinetic analyses, and X-ray structural studies. In addition, two ternary complexes of the enzyme with bound S-adenosyl-L-homocysteine and either dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose or dTDP-3-amino-2,3,6-trideoxy-D-galactose were determined to 1.5 or 1.6 Å resolution, respectively. Taken together, these investigations highlight the important role of His 225 in methyl transfer. In addition, the structural data suggest that the methylation reaction occurs via retention of configuration about the C-3 0 carbon of the sugar.

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The Statistical Analysis of Enzyme Kinetic Data

Cited by 716 publications

References 12 publications

Testing the Sulfotransferase Molecular Pore Hypothesis

Testing the Sulfotransferase Molecular Pore Hypothesis

Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

Probing the catalytic mechanism of a C‐3′‐methyltransferase involved in the biosynthesis of D‐tetronitrose

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