The Behavior of Proteins:

Transcription

1 Mary K. Campbell Shawn O. Farrell Chapter 7 The Behavior of Proteins: Enzymes, Mechanisms, and Control Paul D. Adams University of Arkansas

2 The catalytic activities of many enzymes are regulated by 1. Feedback inhibition (products reactants) 2. Allosteric regulation (allosteric t i inhibitor and allosteric activator) 3. Covalent modification (phosphorylation) 4. Proteolysis (eg. Zymogen) 5. Coenzyme

3 Formation of product inhibits its continued production 1. Feedback Inhibition

4 Aspartate transcarbamoylase (ATCase) feedback inhibition product

5 2. Allosteric regulation Allosteric: Greek allo + steric, other shape Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it these substances change the enzyme s e activity by altering the conformation(s) of its 4 structure Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an allosteric inhibitor allosteric activator

6 Allosteric Enzymes The key to allosteric behavior is the existence of multiple forms for the 4 structure of the enzyme allosteric effector (allosteric inhibitor or allosteric activator): a substance that modifies the 4 structure of an allosteric enzyme homotropic otropic op effects: ects the substrate substateasose also serves esasa a positive (stimulatory) modulator, or activator; e.g., the binding of aspartate (its substrate) to ATCase. (tends to be positive regulator) heterotropic tropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP

7 Allosteric Enzymes Allosteric enzymes do not obey Michaelis-Menten kinetics. Two types of allosteric enzyme systems exist Note: for an allosteric enzyme, the substrate concentration at one-half V max is called the K 0.5 ( 與 Km 有所區隔 ) max 0.5 ( K system: an enzyme for which an inhibitor or activators alters K 0.5 V system V system: an enzyme for which an inhibitor or activator alters V max but not K 0.5

8 Substrate-activity curves for representative allosteric enzymes. (a) The sigmoid curve of a homotropic enzyme, in which the substrate also serves as a positive (stimulatory) modulator, or activator. Note the resemblance to the oxygen-saturation curve of hemoglobin.

9 K system positive modulator (allosteric activator) t negative modulator (allosteric inhibitor) The effects of a positive modulator (+) and a negative modulator ( ) on an allosteric enzyme in which K 0.5 is altered without a change in V max. The central curve shows the substrateactivity relationship without a modulator.

10 V system Substrate-activity curves for representative allosteric enzymes. (c) A less common type of modulation, in which V max is altered and K 0.5 is nearly constant.

11 ATCase (Aspartate transcarbamoylase) Rate of ATCase catalysis vs substrate concentration Sigmoidal shape of curve describes allosteric behavior CTP (allosteric inhibitor); ATP (allosteric activator)

12 ATCase Organization of ATCase catalytic unit: 6 subunits organized into 2 trimers regulatory unit: 6 subunits organized into 3 dimers

13 Two Models Suggest Mechanisms for Cooperative Binding The 1st model: MWC model (concerted model). The 2nd model, the sequential model.

14 MWC model Sequential Model T form R form All subunits switch from T R in unison T R Transition only occur in subuints containing bond ligand

15 The Concerted Model (MWC model) Wyman, Monod, and Changeux The enzyme has two conformations R (relaxed): the active form; binds substrate tightly T (tight or taut): t) the inactive form; binds substrate t less tightly in the absence of substrate, t most enzyme molecules l are in the T (inactive) form the presence of substrate shifts the equilibrium i from the T (inactive) form to the R (active) form in changing from T to R and vice versa, all subunits change conformation simultaneously; all changes are concerted ( 一致 )

16 Sequential Model (Cont d) Main Feature of Model: the binding of substrate induces a conformational change from the T form to the R form the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding sequential model represents cooperativity

17 3. Reversible Covalent modification Phosphorylation The side chain -OH groups of Ser, Thr, and Tyr can form phosphate p esters Phosphorylation by ATP can convert an inactive precursor into an active enzyme protein kinases protein phosphatases

18 Example: Membrane Transport Source of PO 4 is ATP When ATP is hydrolyzed, energy released that allows other energetically unfavorable reactions to take place PO 4 is donated to residue in protein by protein kinases

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20 4. Proteolysis-- Zymogen For some enzymes, an inactive precursor called a zymogen is cleaved to form the active enzyme. Chymotrypsinogen (zymogen, inactive enzyme) synthesized and stored in the pancreas a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) S bonds when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide p from the N- terminal end to give π-chymotrypsin

21 Activation of chymotrypsin Activation of chymotrypsinogen by proteolysis

22 Chymotrypsin -- a serine protease -- Because Ser-195 and His-57 are required for activity, they must be close to each other in the active site -- Catalytic triad: His 57, Asp 102, Ser catalyzes the carboxyl side of aromatic side chains (Tyr, Phe, Trp)

23 Chymotrypsin (Cont d) The active site of chymotrypsin shows proximity of 2 reactive a.a.

24 Active Sites and Transition States Enzyme catalysis an enzyme provides an alternative pathway with a lower activation i energy the transition state often has a different shape than either the substrate(s) or the product(s) True nature of transition state is a chemical species that is intermediate in structure between the substrate and the product. Transition state t analog: a substance whose shape mimics i that t of a transition state In 1969 Jenks proposed that an immunogen would elicit an antibody with catalytic activity if the immunogen mimicked the transition state of the reaction the first catalytic antibody or abzyme was created in 1986 by Lerner and Schultz *(Biochemical Connections, p. 196)

25 Induced fit model

26 Enzyme Specificity Absolute specificity: catalyzes the reaction of one unique substrate to a particular product Relative specificity: i catalyzes the reaction of structurally related substrates to give structurally related products Stereospecificity: catalyzes a reaction in which one stereoisomer is reacted or formed in preference to all others that might be reacted or formed

27 Enzymes can be stereospecific (Specificity where optical activity may pay a role) Asymmetric binding Binding sites on enzymes Binding sites on enzymes must be asymmetric

28 5. Coenzymes Coenzyme: a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction metal ions- can behave as coordination compounds. (Zn 2+, Fe 2+ ) organic compounds, many of which are vitamins or are metabolically related to vitamins (Table 7.1).

29 NAD + /NADH Nicotinamide adenine dinucleotide (NAD + ) is used in many redox reactions in biology. Contains: 1) nicotinamide ring 2) Adenine ring 3) 2 sugar-phosphate groups

30 NAD + /NADH (Cont d) NAD + is a two-electron oxidizing agent, and is reduced to NADH Ni i id i i h d i id i Nicotinamide ring is where reduction-oxidation occurs

31 The B6 vitamins are coenzymes involved in amino group transfer from one molecule to another. Important in amino acid biosynthesis

32 Some important nouns Proenzyme (Zymogen ) : the inactive or nearly inactive precursor of an enzyme, converted into an active enzyme by proteolysis. Holoenzyme: An active, complex enzyme consiting of an apoenzyme and a coenzyme Holoenzyme = apoenzyme +coenzyme (or cofactor) Coenzyme: low molecular weight organic molecules (NAD+, FAD+ ) Cofactor: metal ions, is required for holoenzyme s activity (metalloenzyme) Prosthetic group: the nonprotein component of a conjugated protein, as the heme group in hemoglobin Activation factor: only affect the rate of reaction