Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2007
Lecture Notes: 3 October	© R. Paselk 2007
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Enzyme Kinetics, cont.

Multi-substrate Enzymes

Look at three common and easily understood types. We will use Cleland Nomenclature and "Kinetic mechanism diagrams."

• Ordered Sequential Bi Bi mechanism (two on; two off); Note: A must bind first, Q is released last.
• Ping Pong Bi Bi (one on, one off; one on, one off); Note: have some sort of modified enzyme intermediate (often covalent intermediate)
• Random Sequential Bi Bi (two on; two off); Note: A or B may bind first, P or Q may be released last.

Note that in each case we can predict/explain the pattern of inhibition on the basis of the substrate and inhibitor binding to the same "enzyme form." Thus for the Ordered Sequential mechanism only the first substrate and last product bind to the same form, in this case the free enzyme. Similarly for the Ping pong mechanism the first substrate and last product should be competitive as the both bind the free enzyme. In this case we also see a competitive inhibition between the second substrate and the first product, since they both bind to the E-X complex. The Random Sequential mechanism is a bit more subtle. Here we see across the board noncompetitive since in each case the substrates (and products) can each bind to more than one substrate form, so competitive inhibition will not be possible! (Think of the product as competing with one order of binding but not the other.)

TEMPERATURE AND pH EFFECTS ON ENZYMES (AND PROTEINS)

Temperature profile reflects two underlying phenomena:

• The effects of temperature on rate due to the fact that as temp. increases more molecules will have the energy required to achieve activation energy, E_a. (Review: Arrhenius equation: , and the Maxwell-Boltzman Distribution.)
• The effects of temperature on protein stability: at high temperatures the protein denatures.

Together these effects lead to the plot below where the rising leg is due to activation energy effects (increasing rate) and the falling leg is due to protein denaturation.

pH EFFECTS ON ENZYME RATE

Papain: inflection at pH 4.2 for cys-25 and at pH 8.2 for his-159.

Note that the two legs represent two pH titration curves (rotate the left leg 90 deg. then flip; rotate the right leg 90 deg. counter clockwise and you can see them), with pK's equal to 4.2 and 8.2 respectively. This is a typical example for an enzyme with titratable groups in the active site. Can also have non-symmetrical curves with only one group. And of course can have curves due to denaturation by titration of charged surface and interior side chains.

Zymogens: define and give examples of trypsin/trypsinogen.

Enzyme Catalysis

We will look at catalysis in two types of systems:

• Model systems in organic chemistry to elucidate probable mechanisms of chemical catalysis.
• Example enzymes to demonstrate these mechanisms in enzyme catalysis.

Mechanisms of Chemical Catalysis

Look at some examples of catalysis in model systems (organic chemistry) and how they might operate in enzymes.

Types of Catalysis:

• Acid/Base
  • Specific (H⁺ & OH^- in water)
  • General (Bronsted acid definition: proton donor/acceptor pairs; buffers.)
• Covalent
  • nucleophilic
  • electrophilic
• Proximity/orientation
• Stabilization of Transition State Conformation (Strain/distortion; Charge neutralization)
• Metal/Metal ion.

So how does catalysis work? Recall that the slow step of a reaction is reaching the transition state. Thus if we can find a way to stabilize the transition state (lower E_a) then the reaction rate will be enhanced. Generally we will be looking at three ways to increase rates

1. stabilize transition states
2. increase the concentrations of intermediates
3. use a different reaction pathway.

Pathway Diagrams

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Last modified 3 October 2007