Browsing by Subject "Enzyme kinetics"

Now showing 1 - 3 of 3

Activity and kinetics of microbial extracellular enzymes in organic-poor sands of a south Texas estuary
(2009-08) Souza, Afonso Cesar Rezende de, 1968-; Gardner, Wayne Stanley, 1941-; Pease, Tamara Kaye
The respective kinetics of bacterial leucine aminopeptidase and [beta]-glucosidase activities were investigated to improve understanding of factors controlling activity and hydrolytic capacity in estuarine organic-poor sands. Depth distributions of enzyme activity and bulk organic matter content were determined in sediments of Aransas Bay and Copano Bay Texas, to investigate enzyme dynamics as related to the geochemical properties of the sediment. Vertical profiles of activity in sediment showed that the enzymes were more active at the surface and that the potential hydrolysis rate of leucine aminopeptidase was higher than that of [beta]-glucosidase. Vertical patterns of enzyme activity correlated (weakly) with variations in sediment organic matter (TOC, TN, and carbohydrates) content. Enrichments of sediment samples with monomeric organic compounds and inorganic nutrients did not affect leucine aminopeptidase and [beta]-glucosidase activities in short- and long-term incubations. Enzyme activity was independent of nutrient availability and suggested that microbial communities were not nutrient-limited. Time-course assays of bacterial hydrolysis of TOC, TN, and carbohydrates provided information about how substrate limitation may affect enzyme activity. Positive correlations between bulk TOC and TN content and enzyme activity indicated enzyme dependence on polymeric substrate content. Induction of enzyme activity after sediment enrichments with specific labile compounds confirmed the importance of available organic substrate to enzyme hydrolysis efficiency. A kinetic approach established the occurrence of enzyme inhibition and its effects on enzyme hydrolytic capacity. The addition of a specific-enzyme substrate to sediment samples modified enzyme parameters and indicated that a substrate-reversible type of inhibitor could reduce enzyme hydrolytic capacity. The addition of polyphenol, as a natural inhibitor of enzyme activity, to the sediment resulted in a concomitant reduction of leucine aminopeptidase activity and ammonium regeneration rate, and thus demonstrated a close coupling between enzyme activity and sediment ammonium regeneration. These research results demonstrate the dynamic nature of the hydrolytic enzymes, provide information about the mechanisms of induction and inhibition of activity, and demonstrate some implications of reducing the hydrolytic capacity to organic matter decomposition and nutrient regeneration rates.
Kinetic characterization of the inhibition, excision mechanisms, and fidelity of Hepatitis C Virus RNA-dependent RNA polymerase
(2020-03-03) Villalba, Brian; Johnson, Kenneth A. (Kenneth Allen); Russell, Rick; Fast, Walter L; Dudley, Jaquelin P
NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the Hepatitis C Virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs Mericitabine and Sofosbuvir, which get metabolized to the chain terminators 2’-fluoro-2’-C-methylcytidine-5’-triphosphate and 2’fluoro-2’-C-methyluridine-5’-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, work in our lab has shown that NS5B can efficiently remove chain-terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study I use transient-state kinetics to probe the mechanism of inhibition for nucleoside analogs by directly measuring the rates of incorporation, pyrophosphorolysis, and ATP-mediated excision. I find that while CTP and CTP analogs are readily incorporated, they are efficiently excised. However, UTP is highly resistant to excision, and the 2’-C modifications of UTP serve to further inhibit excision. Furthermore, I use these same techniques to measure the in vitro fidelity of NS5B and uncover mechanisms for maintaining fidelity. The data demonstrate that NS5B exhibits a range of fidelity dependent on the nature of the mismatch. I also identified a slow-pyrophosphorolysis mechanism by NS5B used to further increase fidelity by decreasing k [subscript cat] /K [subscript m]. Together, this work offers insight into how current antiviral therapeutics escape excision, and can aid in the development of new antivirals by furthering our understanding of NS5B.
Phosphatases and prolyl-isomerase in the regulation of the C-terminal domain of eukaryotic RNA polymerase II
(2012-12) Zhang, Mengmeng; Zhang, Yan Jessie; Robertus, Jon D.; Appling, Dean R.; Siegel, Dionicio R.; Fast, Walter L.
In eukaryotes, the first step of interpreting the genetic information is the transcription of DNA into RNA. For protein-coding genes, such transcription is carried out by RNA polymerase II. A special domain of RNA polymerase II, called the C-terminal domain (CTD), functions as a master controller for the transcription process by providing a platform to recruit regulatory proteins to nascent mRNA (Chapter 1-2). The modifications and conformational states of the CTD, termed the 'CTD code', represent a critical regulatory checkpoint for transcription. The CTD, found only in eukaryotes, consists of 26--52 tandem heptapeptide repeats with the consensus sequence, Tyr₁Ser₂Pro₃Thr₄Ser₅Pro₆Ser₇. Phosphorylation of the serines and prolyl isomerization of the prolines represent two major regulatory mechanisms of the CTD. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Understanding how those modifying enzymes recognize and regulate the CTD is important for expanding our knowledge on the transcription regulation and deciphering the 'CTD code'. During my PhD study, I studied the function of CTD phosphatases and prolyl isomerase in the CTD regulation using Scp1, Ssu72 and Pin1 as model regulators. Scp1 and Ssu72 are both Ser5 phosphatases. However, Ssu72 is an essential protein and regulates the global transcription while Scp1 epigenetically silences the expression of specific neuronal genes. Pin1 is a highly conserved phosphorylation-specific prolyl isomerase that recognizes the phospho-Ser/Thr-Pro motif within the CTD as one of its primary substrates in vivo. Among these enzymes, Scp1 is the focal point of this dissertation, as it was studied from different angles, such as enzymatic mechanism (Chapter 3 describes the capture of phospho-aspartyl intermediate of Scp1 as a direct evidence for the proposed two-step mechanism), specific inhibition (Chapter 4 describes the identification and characterization of the first specific inhibitor of Scp1), and its non-active-site contact with the CTD (Chapter 5 describes the structural basis of this contact). These studies are of great importance towards understanding the molecular mechanism of the dephosphorylation process of the CTD by Scp1.