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Allosteric Enzymes
Allosteric: Greek allo + steric, other shape Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it
these substances change the enzymes activity by altering the conformation(s) of its 4structure
Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an
feedback inhibition
Feedback Inhibition
Formation of product inhibits its continued production
ATCase
Rate of ATCase catalysis vs substrate concentration Sigmoidal shape of curve describes allosteric behavior ATCase catalysis in presence of CTP; ATP
ATCase (Contd)
Organization of ATCase catalytic unit: 6 subunits organized into 2 trimers regulatory unit: 6 subunits organized into 3 dimers Catalytic subunits can be separated from regulatory subunits by a compound that reacts with cysteine (phydroxymercuribenzoate)
Note: for an allosteric enzyme, the substrate concentration at one-half Vmax is called the K0.5 K system: an enzyme for which an inhibitor or activators alters K0.5 V system: an enzyme for which an inhibitor or activator alters Vmax but not K0.5
allosteric effector: a substance that modifies the 4 structure of an allosteric enzyme homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP
the binding of substrate induces a conformational change from the T form to the R form the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding sequential model represents cooperativity
Membrane Transport
Source of PO4 is ATP When ATP is hydrolyzed, energy released that allows other energetically unfavorable reactions to take place PO4 is donated to residue in protein by protein kinases
Zymogens
Zymogen: Inactive precursor of an enzyme where cleavage of one or more covalent bonds transforms it into the active enzyme Chymotrypsinogen synthesized and stored in the pancreas a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bonds when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the Nterminal end to give -chymotrypsin
Activation of chymotrypsin
Activation of chymotrypsinogen by proteolysis
Chymotrypsin
A15-unit polypeptide remains bound to -chymotrypsin by a single disulfide bond -chymotrypsin catalyzes the hydrolysis of two dipeptide fragments to give -chymotrypsin -chymotrypsin consists of three polypeptide chains joined by two of the five original disulfide bonds changes in 1structure that accompany the change from chymotrypsinogen to -chymotrypsin result in changes in 2- and 3structure as well. -chymotrypsin is enzymatically active because of its 2and 3structure, just as chymotrypsinogen was inactive because of its 2- and 3structure
What is the mechanism by which the essential amino acid residues catalyze the reaction?
As a model, we consider chymotrypsin, an enzyme of the digestive system that catalyzes the selective hydrolysis of peptide bonds in which the carboxyl group is contributed by Phe or Tyr
Chymotrypsin
Reaction with a model substrate
Chymotrypsin (Contd)
Chymotrypsin is a serine protease
DIPF inactivates chymotrypsin by reacting with serine-195, verifying that this residue is at the active site
Chymotrypsin (Contd)
H57 also critical for activation of enzyme Can be chemically labeled by TPCK
Chymotrypsin (Contd)
Because Ser-195 and His-57 are required for activity, they must be close to each other in the active site Results of x-ray crystallography show the definite arrangement of amino acids at the active site
In addition to His-57 and Ser-195, Asp-102 is also involved in catalysis at the active site
The folding of the chymotrypsin backbone, mostly in antiparallel pleated sheet array, positions the essential amino acids around the active-site pocket
Chymotrypsin (Contd)
The active site of chymotrypsin shows proximity of 2 reactive a.a.
Mechanism of Action of Critical Amino Acids in Chymotrypsin Serine oxygen is nucleophile Attacks carbonyl group of peptide bond
Catalytic Mechanisms
General acid-base catalysis: depends on donation and acceptance of protons (proton transfer reactions) Nucleophilic substitution catalysts- Nucleophilic electron-rich atom attacks electron deficient atom.
same type of chemistry can occur at active site of enzyme: SN1, SN2
Enzyme Specificity
Absolute specificity: catalyzes the reaction of one unique substrate to a particular product Relative specificity: catalyzes the reaction of structurally related substrates to give structurally related products
Stereospecificity: catalyzes a reaction in which one stereoisomer is reacted or formed in preference to all others that might be reacted or formed
example: hydration of a cis alkene (but not its trans isomer) to give an R alcohol (but not the S alcohol)
Asymmetric binding Enzymes can be stereospecific (Specificity where optical activity may pay a role)
an enzyme provides an alternative pathway with a lower activation energy the transition state often has a different shape than either the substrate(s) or the product(s) True nature of transition state is a chemical species that is intermediate in structure between the substrate and the product.
Transition state analog: a substance whose shape mimics that of a transition state In 1969 Jenks proposed that
an immunogen would elicit an antibody with catalytic activity if the immunogen mimicked the transition state of the reaction the first catalytic antibody or abzyme was created in 1986 by Lerner and Schultz *(Biochemical Connections, p. 196)
Coenzymes
Coenzyme: a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction metal ions- can behave as coordination compounds. (Zn2+, Fe2+) organic compounds, many of which are vitamins or are metabolically related to vitamins (Table 7.1).
NAD+/NADH
Nicotinamide adenine dinucleotide (NAD+) is used in many redox reactions in biology. Contains: 1) nicotinamide ring 2) Adenine ring 3) 2 sugar-phosphate groups
NAD+/NADH (Contd)
NAD+ is a two-electron oxidizing agent, and is reduced to NADH Nicotinamide ring is where reduction-oxidation occurs
B6 Vitamins
The B6 vitamins are coenzymes involved in amino group transfer from one molecule to another. Important in amino acid biosynthesis
Pyridoxal Phosphate
Pyridoxal and pyridoxamine phosphates are involved in the transfer of amino groups in a reaction called transamination