Ch7 Enzymes II: Coenzymes, Regulation, Abzymes, and Ribozymes 阮雪芬 NTU

Transcription

1 Ch7 Enzymes II: Coenzymes, Regulation, Abzymes, and Ribozymes 阮雪芬 NTU

2 Enzyme: Coenzyme Partners Vitamins and coenzymes Coenzyme: an organic or organometallic molecule that assists an enzyme. Vitamins: a group of relatively small, organic molecules essential in small amounts in the diet for proper growth and development. Water-soluble: B 2, B 1, B 6, B 12, C Fat-soluble: A, D, E, K 1

3 Characteristics of Vitamins and Coenzymes

4 Characteristics of Vitamins and Coenzymes

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6 Water-soluble and Fat-soluble Vitamins Water-soluble vitamins are usually not harmful. Fat-soluble vitamins may causes serious effects because of accumulation in fat tissue and membranes.

7 Metals as Nutrients Are required for enzyme catalytic action.

8 Allosteric Enzymes Allosteric can be translated to other shapes. Allosterism is the binding or catalytic event occurring at one site influnces the binding or catalytic event at another site. Are influenced by the reversible, noncovalent binding of a signal molecule. Much larger and more complex than nonallosteric enzymes. All have two or more subunits; that is, they are oligomeric. Have catalytic sites (active sites for reaction) and regulatory sites (for binding specific effectors)

9 A Hypothetical Sequence of Reactions Comprising a Metabolic Pathway E 1, E 2, E 3, E 4 and E 5 are regulatory enzymes.

10 Effectors or Modulators Biomolecules that influence the action of an allosteric enzyme. Positive effectors: stimulants to the enzyme. (A in Fig 7.1) Negative effectors: inhibitors to the enzyme. (P in Fig 7.1)

11 Rate Curves for Allosteric Enzymes Sigmoidal Because Michaelis- Menten kinetics are not obeyed, a KM cannot be defined as usual.

12 Rate Curves for Allosteric Enzymes V max is modulated with constant [S] 0.5

13 A Hypothetical Allosteric Enzyme Cooperative and homotropic allosterism (Tetrameric)

14 Kinetic Curve for Allosteric Enzyme

15 A Hypothetical Allosteric Enzyme Heterotropic allosterism (Dimeric) Regulatory subunit Catalytic subunit

16 Models to Describe Allosteric Regulation MWC concerted model Was proposed in 1965 by three French biochemists, Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux. Sequential model Was proposed in 1966 by Daniel Koshland, Jr.

17 MWC Concerted Model

18 Sequential Model A dimer in the RT state is possible Conformational changes

19 MWC and Sequential MWC concerted model Sequential model R and T form are in equilibrium Does not assume initial equilibrium between R and T. The change to the R form is induced by substrate binding All or none RT form is allowed

20 Cellular Regulation of Enzyme Covalent modification of regulatory enzyme Phosphorylation of OH groups in serine, threonine or tyrosine. Attachment of adenosyl monophosphate to a similar OH group. Reduction of cysteine disulfide bonds

21 See p167. Glycogen Phosphorylase

22 Glutamine Synthetase Enzyme + ATP Enzyme + PPi OH O-AMP Inactive

23 Glyceraldehyde-3-phosphate dehydrogenase In Plants Enzyme + AH 2 Enzyme + A S-S SH SH Inactive

24 Activation by Proteolytic Cleavage Zymogen: inactive precursor Is cleaved at one or a few specific peptide bonds to produce the active form of the enzyme. Proteolytic Cleavage An irreversible process and only once in the lifetime of an enzyme molecule

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26 Catalyzes the hydrolysis of peptide bonds on the carboxyl side of large, hydrophobic amino acid residues, such as phenylanine, tyrosine, and leucine. Chymotrpsin

27 Chymotrpsin Is synthesized in the pancreas and secreted into the small intestine. A single polypeptide chain with 245 amino acids residues and cross-linked by five intrachain disulfide bonds

28 Chymotrypsinogen and Chymotrpsin

29 Regulation by Isoenzymes Isoenzymes (Isozymes) Some metabolic processes are regulated by enzymes that exist in different molecular forms. Such mutiple forms All forms demonstrate enzymatic enzyme activity and catalyze the same biochemical reaction, but they may differ in kinetics (different K M and V max ) regulatory properties (different effectors) The form of coenzyme they prefer Their cellular distribution

30 Electrophoresis of the Isozymic Forms of Lactate Dehydrogenase (LDH) LDH Tetramer composed of two possible types of subunits, M and H. M and H are made from two separate genes, are similar in amino acid sequence but can be separated by electrophoresis. M 4 in skeletal muscle H 4 in heart muscle Mixture of five possible forms (M4, M 3 H, M 2 H 2, MH 3, H 4 ) in liver

31 Site-Directed Mutagenesis Early methods Treatment of the protein with chemical reagents that modify amino acid side chains Mutagenization of an organism with ionizing radiation, ultraviolet light, or chemical mutagens. New recombinant DNA procedures

32 Recombinant DNA Technology 1 2 and 3 4 and 5

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35 Antibody and Enzyme Antigen Foreign molecules Antibody and Enzyme Antibodies specifically bind antigen-like molecules in their ground state. Enzyme selectively bind substract molecules in the transition state of a reaction.

36 Abzymes: Catalytic antibodies Reported in 1986 by Peter Schultz and Richard Lerner Catalyze the hydrolysis of esters and carbonates.

37 The Design of an Abzyme

38 Ribozymes Catalytic RNA Some forms of RNA can serve as a biological catalyst was acknowledged by the awarding of 1989 Nobel Prize in Chemistry Sidney Altman Thomas Cech Ribozyme: RNA enzymes Ribonuclease P Self-splicing RNA introns

39 Substrate for Ribonuclease P

40 Self-Splicing RNA Introns T. Cech: Splicing of an intron from pre-rrna was autocatalytic. The RNA cleaved itself without the assistance of protein catalysts

41 Self-splicing of an rrna Precursor

42 Significance of Ribozymes Their use as tools in specific cleavage of RNA Has been designed to catalyze the in vitro cleavage of the RNA of HIV

43 Exercises 7.1 c, i, k, l, m, n