Ch7 Enzymes II: Coenzymes, Regulation, Abzymes, and Ribozymes

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, 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)
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
MichaelisMenten 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
Heterotropic allosterism (Dimeric)
Regulatory subunit
Catalytic subunit
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.
MWC Concerted Model
Sequential Model
• A dimer in the RT state
is possible
• Conformational
changes
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
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
OH
Enzyme + PPi
O-AMP
Inactive
Glyceraldehyde-3-phosphate
dehydrogenase In Plants
Enzyme + AH2
S-S
Enzyme + A
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 five
intrachain disulfide bonds
Chymotrypsinogen and Chymotrpsin
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 KM and Vmax)
regulatory properties (different effectors)
The form of coenzyme they prefer
Their cellular distribution
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.
– 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 Technology
1
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
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7.1 c, i, k, l, m, n
7.4
7.5
7.7
7.9
7.10
7.13