Enzymology

Course unit title

Scientific direction

Scientific code

Faculty

Department (s)

Enzymology

Biochemistry N 004

Life Sciences Center

Institute of Biochemistry

Institute of Biosciences

Total number of credits

Mode of studies

Number of credits

Mode of studies

Number of credits

Lectures

0

Consultations

1

Self-studies

7

Seminars

0

Aims of course

Doctoral students should update knowledge of chemical catalysis, they should understand where, why and how catalysis is needed, what is general-acid-base catalysis, electrostatic catalysis, metal ion catalysis, covalent catalysis. After completion of Enzymology course, students should have essentials of theory of transition state and its application, they should be able to deduce the basic equations of enzyme kinetics and calculate kinetics constants. Students should know about inhibition of enzymes and should distinguish various types of inhibition. They must know pH and temperature dependence of enzyme-catalysed reactions and understand mechanisms of regulation of enzyme activity. Students should understand mechanisms of enzymatic reactions and importance of coenzymes in catalysis. They will learn the methods of enzyme immobilization and kinetic properties.

Main topics

Steady-state kinetics of enzymatic reactions (the Michaelis-Menten equation, validity of steady-state assumption; Van Slyke, Cullen and Briggs, Haldane equations; kinetics of the reversible Michaelis-Menten mechanism; transformation of Michaelis-Menten equation; two intermediates in steady-state kinetics; principle of the King-Altman method, the study of two-substrate two-product reactions; classification of kinetic mechanisms according Cleland).

Inhibition of enzymes (reversible and irreversible inhibition; types of reversible inhibition; Lineweaver-Burk plots for competitive, noncompetive, uncompetitive and mixed-type inhibition; a graphical methods for determining inhibition constants and the interpretation of their meaning).

Measurement of individual rate constants (rapid mixing and sampling techniques; flash photolysis; pulse radiolysis; relaxation methods; analysis of pre-steady state and relaxation kinetics; enzymatic rate constants and rate-determining processes, influence of diffusion).

The pH and temperature dependence of enzyme catalysis (the Michaelis pH functions; pH dependence of V_max and K_M; temperature dependence of rate constants; denaturation and renaturation of enzymes).

Conformational change and allosteric regulation (positive and negative cooperativity; mechanisms of allosteric interactions and cooperativity; quantitative analysis of cooperativity; cooperative binding of oxygen to haemoglobin; allosteric regulation of phosphofructokinase; regulation of glycogen phosphorylase activity by phosphorylation; regulation of metabolic pathways).

Enzymatic reaction mechanisms (proton transfer; nucleophilic substitution reactions; nucleophilic addition to carbonyl group; acyl exchange; phosphoryl exchange; formal hydride or electron transfer; one-carbon chemistry; enolates, enols and enamines; dehydration-hydration and elimination-addition; electrophilic aromatic substitution; concerted reactions; acyl group transfer – proteases, esterases and lipases; aminations; phosphorylation – transfer of phosphoryl group and phosphate esters to water or other acceptors, phosphatases and pyrophosphatases, phosphodiesterases and phosphotransferases.

Coenzymes (mechanisms of pyridoxal-5-phosphate and thiamine pyrophosphate dependent reactions; biotin – structure and role as a carboxyl carrier; heme, FeS and copper in enzymatic reactions; mechanisms of flavin catalysis, structures and functions of nicotinamide coenzymes).

Kinetic properties of immobilized enzymes (kinetic and diffusion control, factors determining the stabilization of immobilized enzymes).

Main literature

• A. Markuckas. Fermentinės katalizės pagrindai. Mokomoji knyga. 2008, „Technologija“.
• A. Markuckas, D. Labeikytė. Fermentinių reakcijų mechanizmai. Studijų kursas Vilniaus universiteto Virtualioje mokymosi aplinkoje.
• A. Cornish-Bowden. Fundamentals of enzyme kinetics. „Wiley Blackwell“, 4^th ed., 2012, p. 510.
• H. Bisswanger. Enzyme kinetics. Principles and methods. „Wiley-VCH Verlag GmbH“, 2^nd ed., 2008, p. 320.

Assessment strategy

Assessment criteria

Examination

Written-oral examination. Answering to two questions. A ten-point system is assessed.

Narimantas Čėnas

Approved by the Council of Doctoral School of Life Sciences Center No (4.10)600000-KT-… on the 14th of October 2021

Chairman dr. Daiva Baltriukienė