ENZYMES
Enzymes serve as catalysts by reducing the free energy, G, of activation of a chemical reaction. Enzymes do this by rearranging electrons in the substrate, creating partial negative and positive charges that favor a reaction. The other ways enzymes do this is by orientating 2 substrate molecules in a way that encourages a reaction between them and by binding to a substrate in such a way that it forces that substrate toward a transition state that favors a reaction. Enzymes play an essential role in metabolism.
Enzymes do not alter the reaction equilibria Keq but rather the rate at which that equilibria is obtained. Many enzynmes have only one substrate, which they bind and then process to produce products. In this case, the reaction is written as follows:
There is an assumption above that the reverse reaction in which E + P recombine to form ES is so small that it can be ignored. In such a case the rate of the reaction, V, can be expressed as V=Kcat[ES] where Kcat is the turnover number, a rate constant that is equal to the number of substrate molecules processed per enzyme molecule each second.
With enzyme catalyzed reactions, the rate, v0, usually increases linearly with substrate concentrations at low concentrations because at low [S] most of the E active sites will be unoccupied so that increasing [S] will bring more E molecules into play and speed up the reaction. At high [S], however, v levels off to a maximal rate called vmax because most of the E's will have their active sites occupied so that the observed rate will depend on the rate at which the bound reactants are converted into products. Thus a graph of initial velocity, V0, on the y axis vs. [S] on the x axis produces a hyperbolic curve which is explained by a very important kinetics equation called the michaelis-menten equation: V0 = Vmax [S] / (Km + [S]) where Vmax is the maximum rate of reaction and where Km is equal to the [S] at which the velocity is 1/2 maximal (the substrate concentration that gives 1/2 Vmax) (units of Km should match that of [S].
A convenient way to evaluate Km and Vmax is to rearrange the MM equation so that 1/v is plotted versus 1/[S]. The result of this is a straight line called the "Lineweaver-Burk plot" . The y intercept will be 1/vmax, the x intercept will be 1/Km and the slope will be Km/Vmax.
Enzymes are usually highly selective and usually catalyze only one particular reaction.
Enzymes have become so efficient that the only factor that limits the reaction rate is no longer the enzyme's intrinsic speed of action but rather the frequency with which the enzyme collides with its substrate. To counter this limiting factor, various enzymes involved in a reaction sequence are often brought together to form a large protein assembly known as a multienzyme complex. Because this allows the product of enzyme A to be passed to enzyme B and so on, diffusion rates need not be limiting, even when the concentrations of the substrates in the cell are very low. Another way that enzymes counter the limiting rate of diffusion is by concentrating particular substrates and their enzymes into membrane enclosed compartmens like the ER which have an extremely small volume.
Regulation of Proteins (Enzymes )
Most enzymes harbor a set of controls that tightly regulate their activity within the cell. Enzymes can be regulated by multiple mechanisms such as their spatial and temporal expression, binding to small-molecule or protein cofactors and posttranslational modification. Thus, attempts to understand functional regulation of an enzyme using an in vitro approach is often misleading. One reason for the irrelevance of in vitro data is the disruption of organelles which lead to the release of activators or inhibitors that artificially affect enzymatic activity.
(1) Feedback inhibition is where an enzyme acting early in a reaction pathway is inhibited by a late product of that pathway. Feedback inhibition can be native in that it can prevent an enzyme from acting or it can be positive in that it stimulates the enzyme's activity. How does feedback inhibition occur? Well, many enzymes have at least 2 different binding sites on their surface. An active site that recognizes the substrates and also a regulatory site that recognizes a regulatory molecule. During feedback inhibition, the binding of an inhibitor at one site on the protein causes the protein to shift to a conformation in which its active becomes incapacitated.
A concept called "linkage" can also help to explain how feedback inhibition can occur. When 2 ligands prefer to bind to the same conformation of an allosteric protein, it follows from basic thermodynamic principles that each ligand must increase the affinity of the protein for the other ligand. For example, if a closed conformation of a protein that binds glucose best also causes a binding site on the protein to fit X better, then the protein will bind glucose more tightly when X is present than when X is absent. Conversely if a shape change caused by glucose binding decreases the affinity of a protein for molecule X, the binding of X must also decrease the protein's affinity for glucose.
A real life example of linkage and feedback inhibition is aspartate transcarbamoylase ("ACTase") which is a rate limiting enzyme of pyrimidine synthesis in bacteria. ACTase is a large complex of 6 regulatory and 6 catalytic subunits. Binding of substrates to the catalytic sites drives ACTase into its catalytically active R state. By contrast, binding of the final products of this pathway, ctyosine triphosphate (CTP) to the regulatory subunits converts the enzyme to an inactive T state from which the substrates dissociate.
(2) Protein Phosphorylation involves the enzyme catalyzed (by a protein kinase) transfer of the terminal phosphate group of an ATP molecule to the hydroxyl (OH) group on a serine, threonine or tyrosine side chain of the protein. The reverse reaction of phosphate removal, dephosphorylation, is catalyzed by a protein phosphatase. The phosphorylation of a protein by a protein kinase can either increase of decrease the protein's activity depending on the site of phosphorylation and the structure of the protein.
Cells contain hundreds of different protein kinases which are responsbile for phosphorylating a different protein or set of proteins. There are also many different protein phosphatases. The state of phosphrylation of a protein and thus its activity depends on the relative activites of the protein kinases and phosphatases that modify it.
In addition to protein kinases and phosphatases, eucaryotic cells use GTP binding proteins (also called GTPases because of the GTP hydrolysis they catalyze). In this case, the phosphate is not attached directly to the protein but rather as a part of the guanine nucleotide GTP which binds to the protein. One such GTP binding protein is the Ras protein which is important for cell signalling. Ras is inactivated by a GTPase activating protein (GAP) which binds to the Ras protein and induces it to hydrolyze its boudn GTP molecule to GDP. Ras stays in this inactive state until it encounters a Guanine nucletoide excahnge factor (GEF) which bind to GDP-Ras and causes ti to release GDP. The empty nucletoide binding stie is immediately filled by a GTP molecule. Thus, in a sense, the roles of GAP an dGEF are analkogous to those of protein phosphatase and protein kinase.
How do Ras like proteins work? Again the answer has to do with conformation changes. An example is provided by the EF-Tu protein. The GTP bound form of EF-Tu forms a tight complex with a rRNA molecules. Only when this GTP is hydorlyzed can the tRNA dissociate. The dissociation is caused by a conformational change along a domain that acts like a latch.
Enzyme Inhibition: is one way that enzymes can be regulated. There are various types of inhibition.
(1) Reversible inhibition: One type of inhibition here is called competitive inhibition: where the inhibitor (I) binds to the active site of the E but even though I looks like S, it can not undergo the catalytic step. Thus for the period of time I occupies the active site, E is unavailable for catalysis and the rate of catalysis of S is reduced. However, competitive inhibition can be overcome by increasing the [S] since as [S] gets very large it outcompetes I for the active site.
A competitive inhibitor will shift the MM curve to the right.
In a Lineweaver-Burk plot, you will see that both competitive and no inhibition lines cross at the same 1/vmax value since the inhibitor has no effect on the value of Vmax. However, the inhibitor straight line will have a higher slope (shifts to the left) in comparison to the no inhibition line. In fact, as you increase [I] the slope will continue to increase. In contrast, if the Ki which is the dissociation constant of the EI complex increases, the slope will decrease. The slope will only increase if the Ki decreases.
(2) Tight binding inhibitors are a special class of competitive inhibitors that can not be analyzed with MM kinetics. These inhibitors bind at very low concentrations and exhibit Ki's on the order of (10-9M).
(2) Irreversible: inhibitors reactive with their target enzyme at low concentrations and form a 1:1 covalent complex. They can not be removed from the enzyme. So Ki does not even exist here. An example is DIPF. A special class of irreversible inhibitor is called a "suicide" inhibitor. The target enyme acts on the inhibitor and converts it to a reactive compound that kills the enyzme's activity. Fluorouracil is an example.