Ch7 Enzymes II:Coenzymes, Regulation, Abzymes,

and Ribozymes阮雪芬

2004/04/23 @ NTU

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

Characteristics of Vitamins and Coenzymes

Characteristics of Vitamins and Coenzymes

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.

Metals as Nutrients

• Are required for enzyme catalytic action.

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)

A Hypothetical Sequence of Reactions Comprising a Metabolic

Pathway

• E1, E2, E3, E4 and E5 are regulatory enzymes.

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)

Rate Curves for Allosteric Enzymes

• Sigmoidal• Because

Michaelis-Menten kinetics are not obeyed, a KM cannot be defined as usual.

Rate Curves for Allosteric Enzymes

• Vmax is modulated with constant [S]0.5

A Hypothetical Allosteric Enzyme (Tetrameric)

Cooperative and homotropic allosterism

Kinetic Curve for Allosteric Enzyme

A Hypothetical Allosteric Enzyme (Dimeric)

Catalytic subunit

Regulatory subunit

Heterotropic allosterism

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.

MWC Concerted Model

Sequential Model

• A dimer in the RT state is possible

• Conformational changes

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

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

Glycogen Phosphorylase

• See p167.

Glutamine Synthetase

Enzyme + ATP Enzyme + PPi

OH O-AMP

Inactive

Glyceraldehyde-3-phosphate dehydrogenase In Plants

Enzyme + AH2 Enzyme + A

S-S SH SH

Inactive

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

Chymotrpsin

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

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

Chymotrypsinogen and Chymotrpsin

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

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

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

Recombinant DNA Technology1

2 and 3

4 and 5

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.

Abzymes: Catalytic antibodies

• Reported in 1986 by Peter Schultz and Richard Lerner– Catalyze the hydrolysis of esters and

carbonates.

The Design of an Abzyme

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

Substrate for Ribonuclease P

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

Self-splicing of an rRNA Precursor

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

Exercises

• 7.1 c, i, k, l, m, n• 7.4• 7.5• 7.7• 7.9• 7.10• 7.13