Award Date

12-1-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Committee Member

Ronald Gary

Second Committee Member

Spencer Steinberg

Third Committee Member

Hong Sun

Fourth Committee Member

Lloyd Stark

Abstract

Kinases are an important class of enzymes involved in the regulation of different cellular processes. The dysfunctional activity, either hyperactivity or inactivity, of kinases has been associated with many types of diseases, making kinases a major therapeutic target. As of 2020, more than 80 kinase inhibitors have been FDA-approved and have revolutionized the treatment for progressive disorders such as cancers and Alzheimer's diseases. However, there is always the possibility of developing severe side effects or resistance to drugs so the search for new therapeutics must continue with efficiency and accuracy.Isothermal titration calorimetry (ITC) is a state-of-the-art technique specialized in detecting the heat absorbed or released from biochemical processes, including enzymatic reactions. Although the primary application of ITC has been in binding studies, the reaction enthalpy detected by ITC can be used to quantify enzyme activity and the efficacy of its inhibitors. Moreover, its ability to use heat as a readout puts ITC at unprecedented advantage against conventional enzyme assays. There has been a number of publications reporting the application of ITC in enzyme kinetics, but kinetic studies of kinases by ITC have been limited. Therefore, T4 polynucleotide kinase (T4 PNK) is proposed as a model system to optimize and validate the ITC analysis conditions for a kinase. T4 PNK typically phosphorylates an oligonucleotide, but a single nucleotide can serve as a substrate if a 3'-phosphate group is present. The T4 PNK-catalyzed conversion of 3'-AMP to adenosine-3',5'-bisphosphate was characterized using the ITC single injection format. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30°C, and the complete Michaelis-Menten analysis was performed with the rates and substrate concentrations derived from the differential power vs. time plot. The Michaelis-Menten constant, KM, was 1.3E-05 M, and the turnover number, kcat, was 8/sec. The effect of inhibitors was investigated using pyrophosphate (PPi). A dose-dependent inhibition of PPi was observed for both KM and kcat of T4 PNK under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3’-AMP were determined to be -25 kJ/mol and 41 kJ/mol, respectively. The reaction enthalpy and kinetic parameters for T4 PNK phosphorylation of 3'-AMP were successfully assessed by single injection ITC analysis. This finding provides a strong foundation to apply the ITC single injection approach to more esoteric kinase systems for which a commercial source of purified enzyme is not readily available.

Keywords

Enzyme Kinetics; Isothermal Titration Calorimetry; ITC; Single Injection Analysis; T4 PNK; T4 Polynucleotide

Disciplines

Biochemistry | Chemistry

File Format

pdf

File Size

3800 KB

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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