FROM PROTEIN SCIENCE TO ENZYMOLOGY

Table of Contents

{1} FROM PROTEIN SCIENCE TO ENZYMOLOGY

{2} Enzymes function...

{3} Selectivity comes from complementary fit and adaptive fit of enzymes to their substrates, remember:

{4} The measure of enzyme function is the reaction rate it catalyzes, remember:

{5} ISSUES FOR THESE LECTURES:

{6} Some Definitions:

{7} Basic units of measurement...

{8} Half saturation?

{9} What equation to use?

{10} What equation...

{11} Definitions cont.

{12} Enzyme Nomenclature and Reaction Types

{13} Enzyme name cont.

{14} Enzyme name cont.

{15} RULES OF ENZYME NOMENCLATURE:

{16} Substrates….

{17} Substrates…

{18} B) Process Names:

{19} Process names...

{20} ENZYME COMMISSION NUMBERS:

{21} Naming conventions for other classes

{22} Naming conventions...

{23} Naming conventions… (EC5. Isomerases)

{24} Naming conventions… (EC.n1.n2.n3.n4)

{25} Naming conventions… (EC.n1.n2.n3.n4)

{26} Some additional n2 examples

{27} ANALYSIS: DETECTION & QUANTITATION

{28} Reasons to use high So relative to Km.

{29} High So in assays...

{30} Issues in Defining Km and kcat

{31} A set of assays looks like a relatively similar set of curves,

{32} @ So = 105 micromolar

{33} @ So = 5 micromolar

{34} Over the range of these So values the linear fitting to the progress curves consistently underestimates the Vi values.

{35} How do you get a better measure of vi?

{36} Jenning & Neiemann...

{37} SUMMARY OF STEPS IN DETERMINING Km and kcat:

{38} Summary...

{39} 1. NON-LINEAR FITTING FOR Km & Vmax:

{40} Non-linear approach...

{41} 2. NON-PARAMETRIC APPROACH:

{42} Non-parametric approach...

{43} 3. LINEARIZATIONS OF THE Michaelis-Menten EQUATION:

{44} Linearization...

{45} Lineweaver-Burk:

{46} EFFECT OF NOISE ON Lineweaver-Burke Plot(using representative samples of simulated data)

{47} Hanes plot transformation (SAME DATA!):

{48} Eadie-Hofstee transformation (SAME DATA!):

{49} These plots show the relative sensitivity with a large data set, however...

{50} “True” values Km = 3, Vmax = 10

Email: wampler@bchiris.bmb.uga.edu

Home Page: http://bmbiris.bmb.uga.edu/wampler/8010

Assignment:

Chapter 11 through section 11.3

Historic References:

Victor Henri (1903) Lois generales de l'action des diastases, Hermann, Paris.

Leonor Michaelis and Maud Menten (1913), Die kinetic der invertin wirkung, Biochem. Z., 19, 333-369.

G. E. Briggs and J. B. S. Haldane (1925), A note on the kinetics of enzyme action, Biochem. J., 19, 339-339.

References:

Arthur R. Schulz (1994) Enzyme Kinetics, Cambridge University Press, Cambridge

Athel Cornish-Bowden (1979) Fundamentals of Enzyme Kinetics, Butterworths, London

International Union of Biochemistry and Molecular Biology, Nomenclature Committee, 1992. Enzyme Nomenclature, Academic Press, New York.

Jenning & Neiemann (1955), JACS 77, 5432-5483.

Dagys et al. (1986), Biochem J. 237, 821-825.

Duggleby (1986), Biochem. J. 235, 613-615.