The material presented on kinetics in the previous section is true for simple enzymes. However in the cell there are many complex enzymes and this is reflected in their quaternary structure. These complex enzymes often contain multiple subunits with active sites. This multiplicity of active sites in a multimeric protein permits the phenomenon of cooperativity between sites, resulting in cooperative kinetics. The model most often used to explore cooperativity in biochemistry textbooks is not an enzyme, but two ligand-binding proteins. A ligand, for the purpose of this discussion, is a small molecule which can reversibly bind to a protein.
Vocabulary:
MYOGLOBIN AND HEMOGLOBIN
Myoglobin is a muscle protein who's function is to store oxygen. The holocomplex consists of a globin protein plus a heme cofactor. Heme is a planar organic molecule - a tetrapyrrole - that has a complexed iron atom at its center. If you look at heme, it has one end that is ionic (carboxyls) whereas the opposite end is hydrophobic.
Myoglobin was the first protein who's 3-D structure was solved, so we have a good idea of how its structure is related to its function.
Features:
Residues exposed on the inside are all non-polar except for 2 histidines. These His residues function to hold the heme ring in place. The heme is tucked into a hydrophobic slot, with the hydrophobic end facing in and the polar end sticking out. The outside surface of the folded protein contains polar residues and a limited number of non-polar residues.
In order to function (i.e., bind oxygen), the iron in the heme must be Fe2+. When heme is bound to the apoprotein, the position of the heme in the small hydrophobic internal cavity protects it from oxidation. The binding of oxygen causes a movement of the Fe2+ and one of the histidines bound to the iron atom; this results in movement of the tertiary structure of the protein.
The reaction is Mb + O2 Mb-O2
The role of myoglobin is to bind oxygen when it is abundant and then to supply oxygen to muscle cells when intracellular concentrations are low. The amount of oxygen bound to myoglobin is usually expressed in terms of the number of oxygen-binding sites which are filled. These units can be percent saturation (which vary from 0 to 100%) or fraction oxygenated (which varies from 0 to 1 and is abbreviated 'Y' in many textbooks). The following relationship (simple hyperbola) exists:
The structure of myoglobin defines its function. The molecular architecture of the protein and the heme define the affinity for O2. The affinity for O2 defines the concentrations at which it will tend to bind O2 and the concentrations at which it will tend to release O2. As the pressure of O2 increases, Mb will tend to bind more; as the pressure of O2 decreases, Mb will tend to bind less. This is important is muscle tissues. When oxygen is relatively abundant, muscle myoglobin binds oxygen. This oxymyoglobin serves as a reserve of oxygen. When muscles become active and begin consuming large amounts oxygen, they can exceed the capacity of the blood to supply oxygen and cellular concentrations of oxygen can plummet. When this happens and cells are close to being anaerobic, the oxygen reserve stored on the myoglobin molecules is released and muscle cell mitochondria can sustain oxidative phosphorylation for a while longer.
You are already familiar with myoglobin, because it is the primary determinant of the color in meats. Myoglobin is found in muscles, the highest concentration being in skeletal muscles that do work. In general, there are two types of muscles; fast-twitch muscles fatigue rapidly and are white, whereas slow-twitch muscles are able to keep contracting for longer times and are red in color. While part of the difference is due to the number of mitochondria, the slow-twitch red muscles have much higher concentrations of myoglobin because of the need for increased O2 storage.
Muscles which are used in locomotion or are actively worked will have greater concentrations of myoglobin. Chicken and turkey [local] are interesting examples. These birds tend not to fly, so the breast muscles used for flying are white meat and low in myoglobin. These birds walk a lot, so the leg and thigh muscles are dark and higher in myoglobin. Pork tends to have about one-fourth of the amount of myoglobin found in beef because hogs don't get as much exercise. Also, myoglobin in muscle tends to increase as an animal ages. The reason veal has a color similar to pork is because myoglobin increases during the life of an animal.
Meat can also take on different colors [local] because of reactions with the iron atom in the heme cofactor. Ferro-heme (Fe2+) with a water in the oxygen binding site is purple. Normally oxygen will bind to form oxymyoglobin which is bright red. During storage or cooking the heme iron can be oxidized to Fe3+, forming metmyoglobin which gives meat its familiar brown color. Reduced myoglobin can also react with nitric oxide (NO) produced in the curing process of preserved meats and form a pink color. You may also sometimes see a pink color in cooked turkey meat; this can be due to differential stability of myoglobin and cytochrome c; during the cooking process turkey meat is heated to 185 °F (85 °C) which is enough to cause denaturation of myoglobin, but the large amounts of cytochrome c in the muscle mitochondria are not denatured until heated to temperatures in excess of 210 °F (100 °C).
Myoglobin is also assayed in clinical blood samples. The presence of myoglobin in blood is as an indication of widespread muscle damage.
Now for a more complex story . . .
Hemoglobin's quaternary structure is alpha2 beta2 (normally called HbA or hemoglobin Alpha-chains are 141 residues each plus 1 heme. Beta-chains are 146 residues each plus 1 heme.
The primary structure is similar to that of myoglobin. It contains 27 invariable residues and about 40 conservative substitutions. The secondary and tertiary structures of the alpha and beta subunits are similar to those of myoglobin. Dimensions: The holoprotein is roughly spherical with a diameter of about 5.5 nm.
Each heme is far enough apart from the others that they don't chemically interact. There are few alpha-alpha or beta-beta contacts between subunits. There are many alpha-beta contacts, mostly hydrophobic.
This graph shows the number of O2 molecules that will be bound to the four subunits of hemoglobin at any given oxygen pressure. The O2 pressure (pO2) is expressed in units of Torr. If hemoglobin is present in the lungs, the O2 pressure is high, approximately 100 Torr. At this concentration each subunit of Hb binds an O2, so a total of 4 moles of O2 are bound per holocomplex. As the blood moves from the lungs to the deep tissues, the O2 pressure drops because the tissues are using the oxygen as a terminal electron acceptor for respiration. As the the pressure drops, O2 molecules start to dissociate from the protein to maintain the equilibrium. By the time the blood reaches capillaries deep inside the body, the oxygen pressure has dropped to about 30±10 Torr and hemoglobin has delivered half of the four molecules it bound in the lungs to the body. Note that the hemoglobin in the blood still retains the capacity to deliver at least an additional O2 molecule if the body is under stress and respiration rates are increased. It is at very low oxygen pressures in muscle tissue that myoglobin assumes its role. As Hb becomes depleted, the low oxygen pressure induces the oxygen bound by Mb to be released, supplying a little more oxygen to keep the muscles working. Once the oxygen in myoglobin is also depleted, the muscle cells are forced to utilize anaerobic glycolysis and lactic acid begins to accumulate.
For hemoglobin, its quaternary structure and the difference in O2 binding lets this exchange work over a much greater [O2] range than possible with myoglobin. When the body is active, extra capacity to transfer oxygen is possible. When the Hb-O2 concentration gets low, then O2 is available from Mb-O2
A model for the sequential oxygen association with hemoglobin.
There are four subunits with a relatively low affinity for O2. Binding one molecule causes a change in structure of the binding subunit that causes an increase in the affinity of the other subunits for O2. The more subunits that bind O2, the more the remaining subunits want to bind O2. This is called cooperativity. The altered position of the Fe2+ upon O2 binding, which changes the position of the bound His residue, releases a cascade of movement. Movement in one subunit causes movement in the other subunits.
Why is the initial binding of O2 with so much less affinity than myoglobin? Because 2,3-bisphosphoglycerate (present at relatively high concentrations in red blood cells) binds between the chains and further distorts the chains of the subunits, causing a lower affinity for O2.
You often hear of people who are asphyxiated from automobile exhaust, defective furnaces or cooking indoors with charcoal. This is a problem caused by carbon monoxide (CO). CO binds very efficiently to hemoglobin. In fact, it is such a better binding ligand than O2 that it is difficult for O2 to displace the CO once it is bound to hemoglobin. Normal blood samples may contain about 3% of the total hemoglobin in the carboxyhemoglobin (CO-Hb) form. Carboxyhemoglobin values above 10% start to cause concern. Values above 20% are considered CO poisoning and values above 50% begin to be fatal. Values in blood of people who smoke are often in the 10 to 15% range.
1. Fetal Hb
The human fetus contains fetal hemoglobins (HbF) instead of adult hemoglobins (HbA). Fetal hemoglobin in formed from two alpha and two gamma subunits. Gamma subunits are produced in the fetus starting at about the eighth week of gestation. At birth, the infant has about 70% of its hemoglobin in the HbF form. By its first birthday, the child normally has less than 1% HbF. These HbF hemoglobins do not bind 2,3-bisphospho glycerate and as a consequence have a higher affinity for O2 than the HbA in the mother's blood. This ensures that the developing fetus is able to acquire O2 from the mother's blood supply. The presence of fetal hemoglobin causes a problem in premature infants because they are less efficient at transferring O2 from the atmosphere to their own tissues.
2. Sickle Cell Hemoglobin
Sickle cell anemia results from a change in the beta subunit primary structure in which Glu6 is changed to Val6 by mutation to form a variant of hemoglobin called hemoglobin S (HbS). Such a mutation causes HbS to polymerize in the red blood cells when partially or fully deoxygenated. The polymerized chains form fibers that distort the cell, causing the characteristic 'sickle' [local] shape. The distorted cells cause problems with capillary circulation [local]. Associated health problems include anemia and tissue injury. Complications include pain in soft tissues and bones, as well as problems with the spleen and kidneys.
Why does the substitution of a Valine for a Glutamate residue cause polymerization? Valine is hydrophobic and makes the surface of the Hb more hydrophobic. Deoxy forms of sickle cell hemoglobins tend to polymerize by hydrophobic associations. This polymer stretches the red blood cells and lyses them. (Visit this site [local] for spectacular electron micrographs.)
Sickle cell anemia is usually only serious in individuals homozygous for the trait. Polymerization is a kinetic process and the rate depends on the concentration of HbS
Homozygotes have all alpha2S2. Heterozygotes have approximately 1 alpha2beta2 : 1 alpha2S2 : 2 alpha2betaS
Cooperative Enzymes
Reflecting on the above material about myoglobin and hemoglobin, myoglobin was simple and bound O2 in a manner similar to the Michaelis-Menten equation for an enzyme reaction. Hemoglobin was more complex in its binding and in its structure. The quaternary structure (alpha2 beta2) caused cooperativity in the binding of oxygen to the subunits.
Some enzymes function this way as well. They have multiple subunits which interact positively or negatively in a cooperative fashion.
Enzymes with this type of behavior do not fit classical Michaelis-Menten kinetics. They are called allosteric. Their behavior can usually be described by a modification of the Michaelis-Menten equation:
The factor "n" describes the degree of cooperativity for the enzyme. For enzymes with simple Michaelis-Menten kinetics, the cooperativity coefficient is 1.0. For hemoglobin oxygen binding, the degree of cooperativity is about 2.8. In general, the cooperativity coefficient (n) is less than or equal to the number of substrate-binding subunits in the enzyme. Because the constant Km was defined in terms of non-cooperative enzymes, a different symbol (S0.5 or K0.5) is usually used to define the substrate concentration at which cooperative enzymes have a velocity that is half of the Vmax.
One feature of cooperative enzymes is that they are more sensitive to changes in substrate concentration in the vicinity of the K0.5 than enzymes displaying Michaelis-Menten kinetics are at their Km values (i.e. the slope of the kinetic response is more steep). For this reason, enzymes that are regulated in biochemical systems are often cooperative enzymes. Regulation by cellular metabolites involves a site, separate from the active site which binds the substrate(s), which binds cellular metabolites indicating whether the particular reaction should be activated or inhibited. Enzymes which respond to the binding of ligands to other sites than the active site are termed 'allosteric' (i.e. other site). Cooperativity is one case of allosteric response; binding of substrate at one active site responds to the binding of another substrate molecule at a different active site.
Allosteric responses may also involve changes in affinity for substrate or Vmax due to the binding of non-substrate molecules at allosteric sites on the enzyme. They usually are regulated by cellular molecules which let them "sense" the need for their products. Such allosteric responses are usually important for cellular regulation.
