Presentation on theme: "Biochemistry SSheng Zhao ( 赵晟 ), Biochemistry and Molecular Department of Medical school in Southeast University WWeb:"— Presentation transcript:
Biochemistry SSheng Zhao ( 赵晟 ), Biochemistry and Molecular Department of Medical school in Southeast University WWeb: http://teaching.ewindup.info/ EEmail: firstname.lastname@example.org or email@example.com QQQ /MSN/Skype/gChat: firstname.lastname@example.org MMobile:18551669724 or 18761413925 Conception, theory, research, and application ——Logic and LIY (Learn It Yourself)
How do enzymes catalyze reactions? ——Stabilize the transition state of a reaction Over the barrier 1.Bond strain 2.Proximity and orientation 3.Proton donors or acceptors (General Acid-base catalysis) 4.Electrostatic catalysis 5.Covalent catalysis Through the barrier 6.Quantum tunneling
1. Bond strain The substrate, on binding, is distorted from the half chair conformation of the hexose ring into the chair conformation by the steric hindrance with amino acids of the lysozyme forcing the change Half chair chair
2. Proximity and orientation Similar reactions will occur far faster if the reaction is intramolecular.
3. Proton donors or acceptors Proton donors (acid) and acceptors (base) may donate and accept protons in order to stabilize developing charges in the transition state. The histidine of the active site accepting a proton from the serine residue
4. Electrostatic catalysis Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial ionic charge interactions) with the intermediate. The tetrahedral intermediate is stabilized by a partial ionic bond between the Zn 2+ ion and the negative charge on the oxygen.
5. Covalent catalysis Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site or with a cofactor. Transient covalent bond Is this similar to another mechanism we learned last time?
6. Quantum tunneling Some enzymes operate with kinetics which are faster than what would be predicted by the classical ΔG‡. In "through the barrier" models, a proton or an electron can tunnel through activation barriers. The tunneling typically enhancing rate constants by a factor of ~1000 compared to the rate of reaction for the classical 'over the barrier' route. This emphasizes the general importance of tunneling reactions in biology.
Components of the enzyme A poenzyme: the protein part of an enzyme Cofactor Inorganic: Metal ions Organic Holoenzyme Simple enzyme Conjugated enzyme Prosthetic group: emphasizes the nature of the binding of a cofactor to a protein (tight or covalent) Coenzymes: additional substance required for enzyme activity A poenzyme ： specificity Cofactor: reaction type
Metal cofactors play a number of roles in transition state stabilization 1. Metals can act as Lewis acids/base (an electron-pair acceptor/donator) 2. Metals can form chelates in enzymes 3. Metals can stabilize charges that develop in the transition state 4. Metals can be important for the structure of the enzyme
Zn functions as a Lewis acid in carbonic anhydrase
Metals can accept and donate electrons in oxidation-reduction reactions
Metals can form chelates in enzymes Chelates are organometallic complexes (I.e. the metal is covalently bound to the enzyme or coenzyme). Heme in hemoglobin
Enzymes need to be active in the right place at the right time Inappropriate expression can lead to uncontrolled growth or cell (and organism) death
How do cells control specific biochemical reactions? Control of substrate availability Removal or conversion of reaction products Control of enzyme levels Control of enzyme location Control of enzyme activity
Control of substrate availability Gluconeogenesis in the liver is regulated by the availability of lactate to the liver. If there is no lactate released into the bloodstream, there will not be any lactate for the liver to convert into glucose. If the substrates of a reaction are not available, the enzyme can’t catalyze their conversion into product.
Removal or conversion of the reaction products Removal or conversion of the reaction products controls the flow of substrates through mass action The conversion of pyruvate into acetyl CoA (or lactate in the case of anaerobic respiration) prevents pyruvate from building up. This increases the rate of glycolysis and inhibits the rate of gluconeogenesis.
Regulation of enzyme levels slow response time energetically expensive maximum activity limited only by relative rates for protein synthesis and degradation generally used for long term changes in enzyme activity
Control of enzyme location Is the enzyme present in the cytoplasm or an organelle, such as the mitochondrion Is the enzyme free in solution or membrane associated? Is the enzyme part of a protein complex?
Regulation of enzyme activity rapid, allows immediate response to stimuli low energy expenditure required maximum activity is limited by amount of enzyme available generally used for short term response
Enzyme activity is regulated by five different mechanisms* 1.Allosteric control 2.Covalent modification 3.Proteolytic activation 4.Stimulation or inhibition by control proteins 5.Monomer ↔ Multimer * changes in enzyme levels due to regulation of protein synthesis or degradation are additional, long-term ways to regulate enzyme activity
Physiology of allosteric enzymes Consider biochemical pathways: - Homotropic regulation, substrate activation activation E1E2 E3 E4 E5 A B C D E F -Heterotropic regulation, end product inhibitionFI.flvFI.flv E1 E2 E3 E4E5 A B C D E F inhibition DEFABCGHIDEFABCGHI F inhibits C->D partially inhibits A -> B
Allosteric regulators do not bind to the active site of the enzyme 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.
Allosteric activators stabilize the high affinity state of the enzyme
Allosteric inhibitors stabilize the low affinity state of the enzyme
Allostery vs Cooperativity Allostery strictly refers to influence of activity by a distant site. Cooperativity indicates that the occupancy of one site in a multisubunit enzyme influences the binding on the others. This is a form of allostery, but is only one manifestation of a general phenomena.
Classic Examples of Allostery Hemoglobin (not an enzyme, Is it familiar?) This was the origin of the "T" and "R" states Aspartate transcarbamoylase. Example of feedback inhibition Types of the cooperativity Positive cooperativity Negative cooperativity
Kinetic Signature of Cooperativity in Enzymes Multisubunit enzymes that exhibit cooperativity show a sigmoidal initial velocity curve in contrast to the hyperbolic curve for independent subunits.
Cooperative enzymes can be allosterically regulated 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. Reaction rate
Noncooperative enzymes can also be allosterically regulated
Covalent modification regulates the catalytic activity of some enzymes Phosphorylation and dephosphorylation （ most common ） Acetylation and deacetylation Methylation and demethylation Adenylation and deadenylation － SH and disulfide bond
Phosphorylation - an example of regulation by reversible covalent modification of the enzyme
Zymogen Pepsinogen Chymotrypsinogen Trypsinogen Procarboxypeptidase Proelastase Prothrombin Fibrinogen Factor VII Factor X Proinsulin Procollagen Procollagenase Active Enzyme Pepsin Chymotrypsin Trypsin Carboxypeptidase Elastase Thrombin Fibrin Factor VIIa Factor Xa Insulin Collagen Collagenase Function protein digestion blood clot formation plasma glucose homeostasis component of skin and bone remodeling processes during metamorphosis, etc. Enzymes involved in protein digestion, blood clotting, and tissue and bone remodeling are synthesized in an inactive conformation, then activated by proteolytic cleavage
Medical Relevance Many diseases are caused by the absence, malfunction, or inappropriate expression of a particular enzyme Enzymes serve as targets for a variety of drugs Enzymes are sometimes administered in the treatment of disease The presence or absence of specific enzymes can be used to diagnose specific diseases
Serum enzymes are commonly used in diagnostic tests for a variety of diseases Myocardial Infarction: Lactate dehydrogenase (H4 isozyme), Aspartate aminotransferase, Creatine kinase Viral hepatitis: Alanine aminotransferase Acute pancreatitis: Amylase, Lipase Liver disease: Alkaline phosphatase, Lactate dehydrogenase (M4 isozyme)
How do serum enzyme levels help in the diagnosis of human disease? “Leak” tissue specific enzymes into the blood system. Different enzyme leak at different time, assaying for the presence of serum enzymes can give information on the time of onset of the disease. (mitochondrial enzymes are generally released after cytoplasmic enzymes)
Drug design: Transition state analogues Enzymes have evolved to recognize the transition state of the reaction they catalyze To design an enzyme inhibitor, we should try to mimic the transition state of the reaction, not the substrates or products
Catalytic Antibodies: Abzymes Molecules which are modified to gain new catalytic activity are called synzymes. An abzyme (from antibody and enzyme), also called catmab (from catalytic monoclonal antibody), A monoclonal antibody with catalytic activity on antigen Abzymes are potential tools in biotechnology, e.g., to perform specific actions on DNA.