Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry

Fall 2008

Lecture Notes: 1 October

© R. Paselk 2008
 
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Myoglobin and Hemoglobin as Example Proteins, cont.

Hemoglobin

Hemoglobin is an alpha-alpha-beta-beta oligomeric protein: its quaternary structure consists of a tetramer of myoglobin like subunits. (text Figure 5-6) The two types of chain are slightly shorter than myoglobin chains (alpha= 141 aa residues, beta= 146 aa residues). There are extensive contacts between an alpha and a beta subunit to give a dimer. The dimers have additional contacts to give the tetramer. Oxygen binding results in a change of conformation in Hb. (text Figure 5-10) The change of conformation affects the binding of oxygen (text Figure 5-11) {oxygen binding is reduced in the "blue" form due to steric hindrance between the oxygen and the heme}.

What about Hb oxygen binding? Obviously more complex. The sigmoid shape (s-shape) of the curve indicates cooperativity (text Figure 5-12, or see below). That is, if one site binds, another is more likely to as well (it cooperates with the first site).

plot of oxygen binding by myoglobin vs. hemoglobin

If the subunits of Hb are fully cooperative (if one subunit binds oxygen they all must bind oxygen, if one releases oxygen they must all release oxygen) then

Hb(O2)4 equilibrium arrow Hb + 4 O2; hemoglobin:oxygen equilibrium expression

and for saturation, substituting as previously with Mb:

hemoglobin:oxygen saturation expression: Y = pO2 to the fourth over pO2 to teh fourth + P50 to the fourth

But this assumption of total cooperativity doesn't work, the curve is too steep.

Hill equation: We can rearrange our equation to find the degree of cooperativity. If we generalize:Hill equation: Y = pO2 to the n over pO2 to tehe n + P50 to the n. Rearranging:

rearranged Hill equation: Y over 1-Y =  pO2 to the n over P50 to the n

In this equation n is the cooperativity, that is the apparent number of fully cooperative sites.

Hill plot: If we take logs of both sides of the Hill equation and plot the results (text Figure 5-14, or see below), the exponent, n, shows up as the slope. Thus we can readily find the apparent cooperativity by plotting saturation vs. oxygen pressure (or concentration). For Hb the slope turns out to be n = 2.8. That is, Hb is partially cooperative - its 4 cooperating subunits only partially cooperate to behave like 2.8 completely cooperative subunits.

Hill Plot: log Y over 1-Y vs log pO2

Note limiting situations at extremes with n = 1. At high concentrations this results because the effective equilibrium is:

Hb(O2)4 equilibrium arrow Hb(O2)3 + O2

In other words it acts like it has a single site (only one site is available at any moment) like myoglobin! At low concentrations of oxygen the opposite effect gives the same result:

HbO2equilibrium arrow Hb + O2

Again, only one site is effectively operating (not enough O2 to fill more than subunit one site at any given time), so again mimics myoglobin.

Allosterism and Regulation

Allosteric ("other site") enzyme or binding proteins are proteins with multiple interacting sites. Allosteric proteins can exhibit one or both of two types of allosterism:

Look at cooperativity/regulation curves for enzymes/binding proteins. Get two families of regulators:

So how to explain the cooperative behavior of allosteric proteins? Need to explain both kinds of effects.

Two important models:

Symmetry Model of Allosterism and Sequential Model of Allosterism (text Figure 5-15).

Symmetry Model of Allosterism (Monod, Wyman & Changeau)

This may be diagramed as in Figure 5.15 of your text, or in a more "classical picture" equilibrium picture showing binding to just one form:

simplified diagram of symmetry model equilibria

 

Can also add binding of effectors to this model: positive effectors bind to R (circles) and shift equilibrium to right, negative effectors bind to T (squares) and shift equilibrium to left.

Sequential Model of Allosterism.

In this model the subunits are each influenced by binding to other subs, but change is step-wise rather than concerted. (text Figure 5-15).

Note that Hb seems to be a combination of both. Some enzymes appear to fit each model.

Introduction to Enzymes

Enzymes are the heart of Biochemistry

Enzymes generally have a cleft for active site, generally <5%of surface: look like pac man (text Figure 6-1). Need large structure to maintain shape etc. with many weak bonds.


Pathway Diagrams

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Last modified 1 October 2008