Download (1) Kinetic Studies -the type of information available from kinetic

(1) Kinetic Studies
-the type of information available from kinetic studies can be seen
from the Table 5.1
(a) Variation of substrate concentration--steady state kinetic
studies support the proposal that one substrate reactions proceed
via the formation and decay of one or more E-S complexes but
cannot give any indication of the sequence of such complexes.
Thus, need additional information from product inhibition,
substrate binding in order to distinguish between mechanisms in
which a ternary complex is formed in an ordered or a random
fashion
(b) Variation of substrate structure--can learn a great deal about
the general features of enzyme active sites by correlating the rates
of reactions with the structures of the substrates
-compare the rates of hydrolysis of a large number of amide
derivatives of amino acids has made it possible to demonstrate
that chymotrypsin has a strong preference for substrates
containing aromatic or bulky hydrophobic R groups cf. elastase
which has a strong preference for substrates containing small
hydrophobic R groups
-the substrate-binding sites of these enzymes must contain
features that account for the observed specificities
-exhaustive studies on specificity of papain towards synthetic
peptide substrates
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-several subsites on the enzyme and that subsite S2 interacts
specifically with the L-Phe side chain
-further work showed that subsite S1' is stereospecific for Lamino acids with a preference for the hydrophobic side chains
of Leu and Trp
(c) Reversible inhibition--study of inhibition of enzymecatalyzed rxns can give information on the structures of active
sites
-most likely explanation for competitive inhibition is that the
substrate and inhibitor bind to the same site on the enzyme
-compare the structure of the substrate and inhibitor and deduce
the essential structural features of these molecules that are
involved in their binding to the active site
-for papain, the tripeptide, Ala-Phe-Arg is a powerful
competitive inhibitor of the enzyme since it occupied the
subsites S3, S2, and S1 and could not therefore undergo
hydrolysis
-competitive inhibitors are used in X-ray crystallographic
studies where it is usually difficult to study the E-S complex
directly
(d) Variation of pH--catalytic activity of many enzymes is
markedly dependent on pH
-many reasons but foremost is the ionization of the amino acid
side chains that are involved in the catalytic mechanism
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-plots of rxn rate versus pH makes it possible to deduce the pKa
values of the side chains involved in catalysis
-caveat: microenvironment of a side chain can shift the pKa of
the side chain by up to four units from the value for the free
amino acid
-approach used to implicate two His side chains as being
involved in the catalytic mechanisms of ribonuclease, later
confirmed by X-ray crystallography
(e) Pre-steady state kinetics--can be used to detect enzymecontaining complexes in a rxn and to determine the rates of
formation and decay of such complexes
-the concn of enzyme more closely approaches that of the
substrates than is the case with steady state studies but usually
requires special techniques to achieve rapid mixing and rapid
detection of the changes occurring
-eg., chymotrypsin catalyzed hydrolysis of p-nitrophenylacetate
-production of p-nitrophenol shows a burst phase (the size
of
which corresponds to approximately 1 mol per mol enzyme)
followed by a slower steady state rate
-consistent with a mechanism where a fast step, corresponding to
the formation of acyl enzyme and release of p-nitrophenol, is
followed by a slow step, corresponding to the rate of hydrolysis of
the acyl enzyme to regenerate enzyme
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(2) Detection of Intermediates--a direct method for obtaining
information about the pathway of a rxn is to detect any
intermediates that may be involved in the rxn
-may be sufficiently stable to be isolated and characterized
-may be inferred to exist from spectroscopic studies
-rates of formation and decay of any presumed intermediate must
be consistent with the overall rate of the rxn
-the rate of breakdown of the acyl enzyme intermediate of
chymotrypsin is very slow at acid pH, so the intermediate can be
isolated (and crystallized) if the enzyme and ester substrate are
mixed and the pH is rapidly lowered
-X-ray crystallographic work has shown that it is the side chain of
Ser 195 that becomes acylated
(3) X-ray Crystallographic Studies--the collection of diffraction
data takes several hours by which time conversion of substrate
to product usually has occurred; however, a number of
approaches used to obtain crystallographic data on the active
site
(i) examine the active complex in the case of a one substrate rxn
when the equilibrium lies very much to one side
(ii) study the enzyme in the presence of a very poor substrate or
competitive inhibitor
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(iii) examine unstable complexes at low temperature where the
rate of decomposition of such complexes will be slowed
(iv) use synchrotron radiation (radiation produced during the
acceleration of charged particles) to reduce data collection time
to seconds
-advantages: direct location of active site and information on
the nature of the side chain involved in the catalytic mechanism;
examine extent of any structural change accompanying the
binding of substrate
-limitations: can only provide a static picture of structure;
danger of differences in structure because of the high ionic
strength conditions required for crystallization of protein-however, many enzymes are active in the crystal state
(4) Chemical Modification of Amino Acid Side Chains--premise
to this approach is simple: if the amino acid side-chain
involved in the catalytic activity is chemically modified, the
enzyme will be inactivated
-identity of modified side chain must be established by standard
structural techniques, e.g., isolating and sequencing a modified
peptide
-problems: chemical modification techniques pose problems
both of design and of interpretation
-requirements: modification rxn must be specific--only one
type of side chain is modified by the reagent (side rxns are
minimal)
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-usually is a problem because many amino acids are
nucleophilic so that an electrophile will react with any number
of side chains, e.g., those of Cys, Lys, His, Tyr, etc.
(a) Applications
(i) Cysteines--Hg has a very high affinity for sulphur so
mercurial reagents should bring about specific modification of
Cys side chains in enzymes
(ii) Tyrosine--activated aromatic ring of Tyr side chain is
susceptible to electrophilic substitution by I2 or by
tetranitromethane
(iii) Acylating Reagents--e.g., iodoacetamide or iodoacetate:
Cys>Tyr>His>Lys
(iv) Arylating Reagents--e.g., 2,4,6-trinitrobenzene sulphonate,
1-fluoro-2,4-dinitrobenzene: Cys>Lys>Tyr>His
-the reactivity of any modification rxn should be checked by
analysis of the modified enzyme, since the reactivity of a side
chain can be considerably influenced by its local environment
-change in the selectivity of certain reagents can be brought
about by changing the pH
-e.g., pH values above 7.0 then
side chain of Cys is extremely reactive toward iodoacetate
because there is a significant proportion of the ionized form CH2-S-, which is the reactive nucleophile
-at pH values below 6, the fraction of the ionized form of the
Cys side chain is much less and the side chain of Met is a more
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reactive nucleophile
-by working at pH 5.6 it was possible to react the Met side
chain with iodoacetate in isocitrate dehydrogenase from pig
heart without any modification of Cys side chains
(v) Super-reactive side chains--sometimes one particular
amino acid in an enzyme is especially reactive because of its
unique environment
-e.g., diisopropylfluorophoshate modifies only the side chain
of Ser 195 and does not react with any of the other 27 Ser side
chains in chymotrypsin or with free Ser
-e.g., Lys 126 from glutamate dehydrogenase (beef liver) is
especially reactive towards 2,4,6-trinitrobenzenesulphonate
(total of 30 Lys in protein)
-in both cases above the super-reactive groups were involved in
the catalytic mechanisms but this is not always the case
(vi) Affinity labelling--to use a modifying reagent which
resembles the substrate and will direct the reagent to the active
site where it will then react with an amino acid in the vicinity of
that site
-e.g., bromohydroxyacetone phosphate acts as an affinity label
for triosephosphate isomerase
-the affinity label binds to the enzyme's active site and the
labile Br atom (activated by the adjacent carbonyl group) can
be displaced by a suitably positioned nucleophilic amino acid
side chain
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-suicide substrates--a substrate that when acted upon by an
appropriate enzyme is converted to product that essentially
irreversibly inactivates the enzyme, usually by covalent
modification
(vii) Interpretation of chemical modification experiments--considerable caution is required because modification of a
particular amino acid side chain which leads to inactivation of
an enzyme does not necessarily mean that the side chain is
directly involved in the catalytic mechanism
-the side chain may be somewhat distant from the active site
but that modification causes a conformational change in the
enzyme leading to loss of activity
Criteria for establishment of side chain involvement in
catalysis
(i) there must be a stoichiometric relationship between the extent
of inactivation and the extent of modification so that complete
inactivation corresponds to the modification of one side chain per
active site
(ii) the addition of substrate or competitive inhibitor must protect
against inactivation since the side chain is no longer accessible to
the modifying reagent
(2) Enzyme Engineering and Design
(a) Site-directed mutagenesis--recombinant DNA technology has led
to the possibility of introducing amino acid replacements at
specified positions in a protein provided:
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(a) gene coding for the protein is available
(b) suitable vector for expression of gene exists
-site-directed mutagenesis is a powerful method for assessing the
importance of particular amino acid side chains in an enzyme
-control experiments necessary to determine that the mutation has
not altered the overall structure of the enzyme which may have
nothing to do with the catalytic mechanism occurring within the
active site of the enzyme
-techniques such as fluorescence spectroscopy, circular
dichroism, and differential scanning calorimetry can be used to
assess secondary structure integrity
-X-ray crystallography of the mutant proteins is also highly
desirable but extremely laborious and time-consuming
(b) Insertion/Deletion.
-add an extra amino acid into the enzyme or remove/delete one
- add or delete a peptide segment
-add affinity tags to aid detection or purification
(c) Fusion Proteins.
-can fuse the genes from two different proteins/enzymes to form a
hybrid enzyme that may boast the best attributes/features of both
enzymes
(d) Novel genes.
-synthesize or transfect novel genes into a cell system or organism
-introduce normal enzymes into defective systems for therapeutics
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(e) Non-Natural Amino Acid Mutagenesis (NNAAM)
(Nowak, M.W. et al., Science 268, 439, 1995)
• used to incorporate an amino acid residue that has novel chemistry not
found in the naturally occurring amino acids
Applications
1. Incorporation of fluorescent reporter groups -- protein dynamics,
conformation, interactions, etc.
2. Positioning of photo-reactive groups for capturing events, trapping
conformations, etc.
3. Providing new chemistry for enzyme reactions to enhance rates or
provide for new reactions
(a) incorporate stronger nucleophiles, electrophiles, more
hydrophobes including more heterocyclic side chains, various acid
and base groups, stronger redox groups
• any specific chemistry that is desired can be incorporated into the
enzyme at the specific site that it is needed
(b) however, must not be too reactive or unstable but capable of
being activated by the appropriate stimulus or chemistry
Method
1. At the protein site (amino acid residue) of interest a stop codon is
incorporated by mutagenesis into the gene
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2. The mutated DNA (gene)
transcription/translation system
is
expressed
in
an
in
vitro
• normally results in a truncated protein
3. If a tRNA bearing an anticodon for the amber stop (a suppressor
tRNA) is added to the system then the amino acid appended to this
suppressor can be "site-specifically" incorporated the protein
• only if the tRNA-NNAA is tolerated by the translation machinery
• suppressor tRNA must not be recognized by any of the normal tRNA
synthetases in the translation system -- so the NNAA is not removed
or replaced by a natural amino acid
4. The tRNA-NNAA must be chemically synthesized
• tRNA from yeastPhe with specific base changes is well suited for the
E. coli or rabbit reticulocyte lysate translation systems
(f) Disulfide bonds.
-decrease the number of unfolded conformations by introducing novel
disulfide bridges which will increase the stability of the newly
engineered enzyme
-the longer the loop between the 2 Cys residues the more restricted is the
unfolded polypeptide
-must be introduced in the proper location or else strain will be present in
the new structure which will decrease the stability of the protein's
structure
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