Oculoplastic Surgery

Leatherbarrow, Brian

doi:10.1055/b-006-161114

Cited by 16 publications

(34 citation statements)

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“…Initial rates were obtained over the course of 1 min. The data were fit to the Michaelis–Menten equation using OriginPro 8 (OriginLab) for the orthologs and the mutants of Ser295, Trp134, and Tyr139 or using the equation for an ordered, sequential, two-substrate mechanism (eq 1) 18 using GraFit 7 19 for 17X-PTDH, R301A, and R301K.In this equation A represents the first substrate to bind (NAD + ) and B represents the second substrate (phosphite). K A and K B are K m,NAD and K m,PT , respectively.…”

Section: Methodsmentioning

confidence: 99%

Investigation of the Role of Arg301 Identified in the X-ray Structure of Phosphite Dehydrogenase

et al. 2012

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Phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri catalyzes the nicotinamide adenine dinucleotide-dependent oxidation of phosphite to phosphate. The enzyme belongs to the family of d-hydroxy acid dehydrogenases (DHDHs). A search of the protein databases uncovered many additional putative phosphite dehydrogenases. The genes encoding four diverse candidates were cloned and expressed, and the enzymes were purified and characterized. All oxidized phosphite to phosphate and had similar kinetic parameters despite a low level of pairwise sequence identity (39–72%). A recent crystal structure identified Arg301 as a residue in the active site that has not been investigated previously. Arg301 is fully conserved in the enzymes shown here to be PTDHs, but the residue is not conserved in other DHDHs. Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ∼100-fold decrease in kcat and an almost 700-fold increase in Km,phosphite for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher kcat than the parent PTDH, and a more modest increase in Km for phosphite (nearly 40-fold). Given these results, Arg301 may be involved in the binding and orientation of the phosphite substrate and/or play a catalytic role via electrostatic interactions. Three other residues in the active site region that are conserved in the PTDH orthologs but not DHDHs were identified (Trp134, Tyr139, and Ser295). The importance of these residues was also investigated by site-directed mutagenesis. All of the mutants had kcat values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

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Section: Methodsmentioning

confidence: 99%

Investigation of the Role of Arg301 Identified in the X-ray Structure of Phosphite Dehydrogenase

et al. 2012

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“…Anisotropy was calculated using the equations as described (23). Observed anisotropy data were fitted to obtain dissociation constants using GraFit fitting software (24). …”

Section: Methodsmentioning

confidence: 99%

PcrA Helicase Tightly Couples ATP Hydrolysis to Unwinding Double-Stranded DNA, Modulated by the Initiator Protein for Plasmid Replication, RepD

Slatter¹,

Thomas

Webb³

2009

Biochemistry

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The plasmid replication initiator protein, RepD, greatly stimulates the ability of the DNA helicase, PcrA, to unwind plasmid lengths of DNA. Unwinding begins at oriD, the double-stranded origin of replication that RepD recognizes and covalently binds to initiate replication. Using a combination of plasmids containing oriD and oligonucleotide structures that mimic parts of oriD, the kinetics of DNA nicking and separation have been determined, along with the coupling ratio between base separation and ATP hydrolysis. At 30 °C, the rate of nicking is 1.0 s−1, and translocation is ∼30 bp s−1. During translocation, the coupling ratio is one ATP hydrolyzed per base pair separated, the same as the value previously reported for ATP hydrolyzed per base moved by PcrA along single-stranded DNA. The data suggest that processivity is high, such that several thousand base-pair plasmids are unwound by a single molecule of PcrA. In the absence of RepD, a single PcrA is unable to separate even short lengths (10 to 40 bp) of double stranded DNA.

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“…All experiments were carried out in triplicate and the kinetic parameters of the reactions catalysed by COXMn and GOX were determined using the nonlinear regression program Grafit32 developed by Leatherbarrow (2000).…”

Section: Discussionmentioning

confidence: 99%