State Kinetic Analysis Research Articles

Investigating the Kinetic Effects of the Allosteric Interaction Between Newly‐Discovered Dynamic Hydrogen‐Bonded Residues and the HINT1 Active Site

Histidine Triad Nucleotide binding proteins (HINTs) are an ancient class of enzyme notable for their acyl‐AMP hydrolase and phosphoramidase activity. This class of protein is of research significance due to its involvement in antiviral prodrug activation, as well as its natural role in opioid tolerance. Previously, the Wagner Lab has published the substrate specificity profile, full kinetic mechanism, and kinetics assay for the HINT isozyme HINT1. However, the active site dynamics of this enzyme have yet to be elucidated.Recent insights into the structural dynamics of HINT enzymes found via CPMG‐NMR and discovered by the Wagner Lab have revealed a potential site of regulation for the kinetic rates of HINT1. We hypothesize that a subtle allosteric interaction exists between the active site of HINT1 and the previously unrecognized distant site through a channel of conserved waters, which plays a key role in the active site dynamics and kinetics.Thus, with the goal of investigating the kinetic properties of this potential allosteric interaction, two genetic mutants of the enzyme HINT1 were produced to eliminate or alter the interactions between the highly dynamic residues Q62 and E100. Both steady‐state and stopped flow pre‐steady state kinetic analyses were performed on the mutants to determine any significant changes to their kinetics. Furthermore, circular dichroism analysis of both mutants was performed to ensure that both mutants were structurally sound, with no significant enzyme misfolding.Through these analyses, it was found that the kcat and Km of the HINT1 mutants were altered significantly and predictably through the pair of successive mutations. Through process of elimination, these findings informed us that these mutations to the dynamic residue Q62 must be altering the rate of intermediate hydrolysis at the HINT1 active site, a site previously thought unlikely to be altered.Support or Funding InformationPartial funding of this study by the University of Minnesota Office of Undergraduate Research is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Physical Parameters on DeNOx Conversion in Selective Catalytic Converter Used in Diesel Vehicles

The Urea SCR system is a promising approach to reduce NOx in order to meet stringent limits on Euro 1V and Euro V standards. Apart from thermodynamic properties (temperature, pressure,heat and mass transfer), the cell geometry of SCR also got significant role in reduction of NOx. The current study focuses on the calculation of NOx conversion by varying the Open Frontal area of monolith, volume of monolith, cell density thereby to choose best cell geometry which will result in maximum DeNOx efficiency. It has been found that as the cell density increases the NOx conversion efficiency also increases. In the current analysis, a cell density varying from 200 CPSI to 400 CPSI is considered. One dimensional steady state and transient kinetic analysis are carried out using AVL BOOST software. The monolith volume is varied from 0.002m3 to 0.008m3 and the effects on DeNOx efficiency are discussed. The Open frontal area of SCR catalyst also been varied, and the effects on NOx conversion is studied. It has been found that as the cell density, monolith volume increases, the NOx conversion efficiency also increases, where as it decreases with increase in Open frontal area. The results are validated through experimental results obtained from the literature.

Werner's syndrome protein limits the error‐prone 8‐oxo‐G lesion bypass activity of human DNA polymerase kappa by promoting the error‐free bypass

Oxidative stress induced DNA damage plays a key role in cancer and aging. 7,8‐dihydro‐8oxo‐2′‐deoxyguanosine (8‐oxo‐dG), produced during DNA oxidation is highly mutagenic and known to block replication. Previous studies show that the human DNA polymerase kappa (hpol κ) bypasses 8‐oxo‐dG by preferentially inserting dATP opposite the lesion. Recent studies indicated that hpol κ coordinates with Werner's syndrome protein (WRN) during the 8‐oxo‐dG bypass. Our data show that WRN stimulates the 8‐oxo‐dG bypass activity of hpol κ by enhancing the correct base insertion opposite the lesion and diminishing the efficiency of dATP mispair insertion. Kinetic analysis reveals that in the presence of WRN, hpol κ inserts the dCTP opposite 8‐oxo‐dG ~ 1.7‐fold more efficiently than dCTP opposite dG. Currently we are investigating the fidelity and catalytic efficiency of hpol κ bypass of 8‐oxo‐dG in the presence of WRN using pre‐steady state kinetic analysis and mass spectrometric detection of polymerization products. WRN may function in coordination with hpol κ to limit the error‐prone bypass of 8‐oxo‐dG, which could suppress the mutagenicity associated with hpol κ and prevent genomic instability. This work is supported in part by USPHS R00 GM084460 (R.L.E).

Control and Function of Translesion DNA Polymerases

Not only is E. coli's translesion DNA polymerase DinB (DNA pol IV) a much better polymerase when copying over N2‐furfuryl‐dG than over ordinary dG, but it is also remarkably proficient at extension, suggesting a biological role of carrying out relatively accurate DNA synthesis over common class(es) of N2‐dG adducts that are common to all domains of life. The products of the umuD and recA genes control functions of DinB as well as those of UmuC (DNA pol V and pre‐steady state kinetic analyses are offering insights into the nature of this control. A mutant DinB generated by altering a evolutionarily conserved tyrosine is capable of insertion opposite the lesion, but cannot complete TLS, stalling three nucleotides after an N2‐furfuryl‐dG adduct. Expression of this variant transforms a bacteriostatic DNA damaging agent into a bactericidal drug, resulting in profound toxicity even in a dinB+ background. Our discovery of physical and genetic interactions between DinB and NusA, a component of elongating RNA polymerase, indicates a previously unrecognized interaction between transcription and TLS that affects several aspects of DNA repair and mutagenesis.