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).

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 Hb + 4 O₂;

and for saturation, substituting as previously with Mb:

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:. Rearranging:

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.

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

Hb(O₂)₄ 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:

HbO₂ 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:

• Homotropic: this is where the sites are identical, and each sites is allosteric to the others. This is like the cooperative interactions seen in oxygen binding by hemoglobin - four (essentially) identical oxygen binding sites interacting with each other allosterically.
• Heterotropic: this is where binding at one kind of site affects the binding at a second kind of site. This occurs in the regulation of oxygen binding in hemoglobin by BPG (BisPhosphoGlycerate = DPG in older literature). BPG affects the binding of oxygen by all of the oxygen sites. [Hb overheads]

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

• Positive (+) effectors or activators. These shift the binding constant so the binding takes place at lower concentrations, or increases the rate of an enzyme at a given concentration. Notice that (+) effectors decrease cooperativity.
• Negative (-) effectors (sometimes called inhibitors, but we will reserve the term inhibitor for classical enzyme inhibitors which work on non-allosteric proteins): decrease binding, so concentration must be increased to give the same level of activity. Notice that (-) effectors increase cooperativity.

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

• Homotropic allosterism as occurs with O₂ binding in Hb.
• Heterotropic regulation of O₂ binding, as occurs with BPG in Hb (text Figure 5-17).

Two important models:

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

Symmetry Model of Allosterism (Monod, Wyman & Changeau)

• Allosteric protein an oligomer of protein subunits that are symmetrically related;
• Protomers can exist in two conformations (designated T[ense]: low affinity for substrate, & R[elaxed]: high affinity for substrate) that are in equilibrium whether or not ligand is bound;
• Ligand can bind to protomers in either conformation, but conformational change alters affinity for the ligand;
• The molecular symmetry of the protein is conserved during conformational change: the protomers must therefore change in a concerted manner.

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:

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

• protein based catalysts (for us RNA based catalysts are Ribozymes)
• enormously effective catalysts: typically enhance rates by 10⁶ to 10¹² fold
• operate under mild conditions: 0 - 100 °C (or perhaps even 300+ °C for some bacteria in deep ocean), 20 -40 °C for most organisms; and low pressures (atmospheric)
• very specific: generally catalyze reaction for a very restricted group of molecules, sometimes for a single naturally occurring molecule of a single chirality.
• Recall that catalysis operates by lowering the activation energy (E) required to reach the transition state, generally by creating an alternate reaction mechanism or pathway. (text Figure 6-3

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