ch7 enzymes ii: coenzymes, regulation, abzymes, and...
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Ch7 Enzymes II:Coenzymes, Regulation, Abzymes,
and Ribozymes阮雪芬
2004/04/23 @ NTU
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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: B2, B1, B6,B12, C• Fat-soluble: A, D, E, K1
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Characteristics of Vitamins and Coenzymes
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Characteristics of Vitamins and Coenzymes
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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.
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Metals as Nutrients
• Are required for enzyme catalytic action.
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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, noncovalentbinding of a signal molecule.
• Much larger and more complex than nonallostericenzymes.
• 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)
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A Hypothetical Sequence of Reactions Comprising a Metabolic
Pathway
• E1, E2, E3, E4 and E5 are regulatory enzymes.
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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)
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Rate Curves for Allosteric Enzymes
• Sigmoidal• Because
Michaelis-Menten kinetics are not obeyed, a KM cannot be defined as usual.
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Rate Curves for Allosteric Enzymes
• Vmax is modulated with constant [S]0.5
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A Hypothetical Allosteric Enzyme (Tetrameric)
Cooperative and homotropic allosterism
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Kinetic Curve for Allosteric Enzyme
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A Hypothetical Allosteric Enzyme (Dimeric)
Catalytic subunit
Regulatory subunit
Heterotropic allosterism
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Models to Describe AllostericRegulation
• 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.
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MWC Concerted Model
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Sequential Model
• A dimer in the RT state is possible
• Conformational changes
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MWC and Sequential
RT form is allowedAll or none
Does not assume initial equilibrium between R and T. The change to the R form is induced by substrate binding
R and T form are in equilibrium
Sequential modelMWC concerted model
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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
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Glycogen Phosphorylase
• See p167.
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Glutamine Synthetase
Enzyme + ATP Enzyme + PPi
OH O-AMP
Inactive
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Glyceraldehyde-3-phosphate dehydrogenase In Plants
Enzyme + AH2 Enzyme + A
S-S SH SH
Inactive
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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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Chymotrpsin
• Catalyzes the hydrolysis of peptide bonds on the carboxyl side of large, hydrophobic amino acid residues, such as phenylanine, tyrosine, and leucine.
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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 fiveintrachain disulfide bonds
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Chymotrypsinogen and Chymotrpsin
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Regulation by Isoenzymes
• Isoenzymes (Isozymes)– Some metabolic processes are regulated by enzymes
that exist in different molecular forms. Such mutipleforms
• All forms demonstrate enzymatic enzyme activity and catalyze the same biochemical reaction, but– they may differ in kinetics (different KM and Vmax)– regulatory properties (different effectors)– The form of coenzyme they prefer– Their cellular distribution
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Electrophoresis of the IsozymicForms 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.
– M4 in skeletal muscle– H4 in heart muscle– Mixture of five possible forms
(M4, M3H, M2H2, MH3, H4) in liver
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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
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Recombinant DNA Technology1
2 and 3
4 and 5
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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.
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Abzymes: Catalytic antibodies
• Reported in 1986 by Peter Schultz and Richard Lerner– Catalyze the hydrolysis of esters and
carbonates.
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The Design of an Abzyme
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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
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Substrate for Ribonuclease P
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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
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Self-splicing of an rRNA Precursor
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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
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Exercises
• 7.1 c, i, k, l, m, n• 7.4• 7.5• 7.7• 7.9• 7.10• 7.13
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