|Lecture Notes: 22 February
© R. Paselk 2006
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]
Of course for any of these effects to exist, must see some degree of protein flexibility.
- Atomic fluctuations (10-15- 10-11 sec; 0.001 - 0.1 nanometer) Myoglobin example. (Figure 4.40 - show motions possible)
- Collective motions of covalently linked atoms, from aa R-groups to domains (10-12 - 10-3 sec; 0.001 - >0.5 nanometer)
- Triggered conformational changes: in response to ligand binding, covalent modification etc.
- How do we know about the mobility of protein structures?
- X-ray diffraction studies of proteins with and without ligand bound
- NMR (phe, his ring protons/carbons show up on edges of signal envelope)
- Antibody binding: make antibodies to normally interior aa residues, over time protein ppt forms as interior groups momentarily exposed.
Look at cooperativity/regulation curves for enzymes/binding proteins. (Fig. 5.21) 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.
Two important models
Symmetry Model of Allosterism and Sequential Model of Allosterism.
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 in simplified form as in Figure 5.22a of your text, or in a more "classical picture":
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. (Figure 5.22b, p 152)
Note that Hb seems to be a combination of both. Some enzymes appear to fit each model. (Figure 5.23, p 153)
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.
Enzymes generally have a cleft for active site, generally <5%of surface: look like pac man. Need large structure to maintain shape etc. with many weak bonds.
Six Classes of enzymes
- Oxidoreductases - redox reactions
- Transferases - group transfer reactions
- Hydrolases - hydrolysis reactions
- Lyases - lysis generating a double bonds (non-oxidative elimination reactions)
- Isomerases - isomerization reactions in a single moleculoe
- Ligases - join (ligate) two substrate molecules
Look at major aspects of enzyme study:
- Molecular mechanisms of catalysis
- Kinetics, including review
Last modified 22 February 2010