Biochem 153A: Week 5 Discussion A Biochemistry Love Story -- An Enzyme Meets Its Substrate



Quote for the Week: "Why one cell becomes bone and another brain, one normal and another cancerous, are questions still without final solutions because we know too little about our enzymes!" -A. Kornberg

I. The Love Story -- Classical Enzyme+Substrate Reaction Kinetics


Objectives: Understand the concepts of active site, initial velocity, transition state, rate-limiting step, turnover number, Km, Vmax, and steady-state within the context of enzymatic reactions; Know how to calculate turnover numbers; Understand how initial velocity plots are generated from experimental data.

II. Michaelis-Menten Kinetics

Objectives: Understand the assumptions and premises used in setting up the derivation of the Michaelis-Menten equation; Be able to derive the Michaelis-Menten equation; Know how to use the Michaelis-Menten equation.
Questions: 1) The Km for p-nitrophenyl phosphate is 5 X 10-6 M in the reaction catalyzed by alkaline phosphatase at pH 8 and 37C. What is the initial velocity that would be observed if the p-nitrophenyl phosphate concentration is 2 X 10-5 M?

2) The hydrolysis of pyrophosphate to orthophosphate is important in driving forward biosynthetic reactions such as the synthesis of DNA. This hydrolytic reaction is catalyzed in e. coli by a pyrophosphatase that has a mass of 120 kDa and consists of six identical subunits. For this enzyme, a unit of activity is defined as the amount of enzyme that hydrolyzes 10mmoles of pyrophosphate in 15 minutes at 37C under standard assay conditions. The purified enzyme has a Vmax of 2800 units per mg of enzyme.

(a)How many moles of substrate are hydrolyzed per second per mg of enzyme when the substrate concentration is much greater than Km?
(b)How many moles of active site are there in 1 mg of enzyme? (Assume that each subunit has one active site, and remember that 1 mole = MW in Da).
(c)What is the turnover number of the enzyme?

III. The Equations (other Famous Couples in Biochemistry)


Objectives: Understand the differences and advantages between Michaelis-Menten, Lineweaver-Burke, and Eadie-Hofstee plots; Know how to plot data and get data (Km and Vmax) from these plots.

IV. Types of Inhibition


Objectives: Realize that all of the examples of inhibition (Competitive, Noncompetitive, and Uncompetititve) can be generated by just remembering the cartoons which we generated in discussion; Understand plots and Ki for each inhibitor; Be able to graph enzyme activity in the presence of inhibitors and determine the type of inhibition.

Consider the case of Ellen, Scott, Carla, Nadine, and Ursula.

Problems: 1) and 2) we did during discussion.
3) You have isolated a new compound, joebruin, and would like to know how it affects the reaction on p-nitrophenylphosphate by alkaline phosphate. The rate of the reaction mixtures of various substrate concentrations are below: 


[p-nitrophenylPi]	reaction rate		reaction rate with joebruin

	1 mM		1.6 mM/sec		1.2 mM/sec

	2.5		4.0			1.4

	5.0		6.5			2.5

	7.5		7.5			3.9

	10.0		8.0			5.0

	15.0		12.4			7.4

	20.0		14.8			8.2

	25.0		15.1			11.5

	30.0		15.2			12.6

	40.0		15.2			15.1

	
--What type of inhibitor is joebruin?

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