Protein Enzymes RNA All enzymes are proteins except some RNAs and not all proteins are enzymes
What is a substrate? –A substrate is the compound that is converted into the product in an enzyme catalyzed reaction. –For the reaction catalyzed by aldolase, fructose 1,6-phosphate is the substrate.
Enzyme Nomenclature Oxidoreductases(EC Class 1) – Transfer electrons (RedOx reactions) Transferases(EC Class 2) –Transfer functional groups between molecules Hydrolases(EC Class 3) –Break bonds by adding H 2 O Lyases(EC Class 4) –Elimination reactions to form double bonds Isomerases(EC Class 5) –Intramolecular rearangements Ligases(EC Class 6) –Join molecules with new bonds
How do enzymes work? Free energy tells us nothing about the rate at which the reaction occurs Free Energy ( G) Enzymes stabilize the transition states of reactions
The active site of the enzyme contains functional groups that stabilize the transition state of the reaction Active site of chymotrypsin
The active site binds the substrates and positions them in the proper orientation for the reaction to occur. The active site contains chemical groups that stabilize the transition state of the reaction. The active site determines the specificity of the enzyme (i.e. it determines whether a particular substrate is bound and whether a particular product is made). What does the active site do?
Characteristics of active sites The active site takes up a small part of the total volume of the enzyme The active site is 3-dimensional and is generally found in a crevice or cleft on the enzyme The active site displays highly specific substrate binding The active site is responsible for whether there is ordered or random binding of substrates and release of products
Understanding K m Km is a constant derived from rate constants Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S, because at equilibrium, Reversible reaction, dissociation constant is So small Km means tight substrate binding; high Km means weak substrate binding. Km equals to the substrate concentration at which v=1/2vmax k1k1 k -1
Understanding V max The theoretical maximal velocity V max is a constant V max is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach V max would require that ALL enzyme molecules are tightly bound with substrate V max is asymptotically approached as substrate is increased
Enzyme Inhibition Enzyme can be inhibited by inhibitors. Inhibitors are tools to scientists to understand enzymes. Inhibitors are also in many cases pharmaceutical reagents against diseases; Inhibitors inhibit enzyme function by binding with enzymes. The binding reaction can be either reversible or irreversible; Reversible inhibitors associate with enzymes through non-covalent interactions. Reversible inhibitors include three kinds: –Competitive inhibitors; –Non-competitive inhibitors; –Un-competitive inhibitors Irreversible inhibitors associate with enzymes through covalent interactions. Thus the consequences of irreversible inhibitors is to decrease in the concentration of active enzymes.
Summary of Classes of Inhibitors Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition. Mixed inhibition-when the dissociation constants of (I) to E and ES are different. The inhibition is mixed.
The catalytic mechanism is conserved among members of the serine protease family
Enzyme activity is regulated by four different mechanisms* (1) Allosteric control (2) Covalent modification (3) Proteolytic activation (4) Stimulation or inhibition by control proteins *changes in enzyme levels due to regulation of protein synthesis or degradation are additional, long-term ways to regulate enzyme activity
Allosteric enzymes: some definitions 1. Allosteric = “other site” other than active site 2. Regulatory molecules called, effectors, modulators, regulatory molecules 3. Homotropic regulation: regulation by substrate at active site 4. Heterotropic regulation: regulation by molecule NOT substrate ( end products), at allosteric site 5. Few enzymes are allosteric 6. Allosteric enzymes DO NOT exhibit M-M kinetics
Cooperative enzymes can be allosterically regulated Reaction rate
Activation or inhibition of an enzyme ’ s activity due to binding of an activator or inhibitor at a site that is distinct from the active site of the enzyme.
What it means: Allosteric inhibitors bind to a site outside the active site of the enzyme, but affect the ability of the active site to catalyze the reaction. In this analogy, the active site is that part of Monkey King that picks up and shells a peanut. In other words, the active site of Monkey King is his hands. The inhibitor molecule and regulatory site are represented by the blindfold and Monkey King ’ s head, respectively. The binding of inhibitor (putting the blindfold over Monkey King ’ s head) reduces the rate at which Monkey King can find peanuts. Since the rate at which Monkey King finds peanuts corresponds to the K m of the enzyme, the effect of putting the blindfold on Monkey King is to increase the K m of the enzyme (Monkey King). Note that this is only true for this specific example. Allosteric inhibitors and activators can affect the affinity of the enzyme for the substrate (K m ), the rate at which the enzyme converts bound substrate into product (k cat ), or both the K m and k cat.
Allosteric regulators shift the substrate dependence curve Reaction rate In the above plot, the allosteric activator decreases the K m of the enzyme, while the allosteric inhibitor increases the K m of the enzyme.
Noncooperative enzymes can also be allosterically regulated
What is an isozyme? (1) Isozymes are physically distinct forms of the same enzyme. (2) Isozymes may differ from each other by differences in their amino acid sequences or by the presence of different posttranslational modifications in each isozyme. (3) The relative abundance of different isozymes varies for different tissues. The ability to control which isozymes are expressed in a particular cell allows each cell to adjust the enzyme activity based on the specific conditions that exist in the cell.
Lactate Dehydrogenase is composed of four monomers Each monomer can be either heart or muscle type Five different isozymes of lactate dehydrogenas e exist: H4, H3M, H2M2, HM3, and M4
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