Enzyme catalytic strategies and regulation

ADVANCED BIOCHEMISTRY Enzyme: Catalytic Strategies and Regulation Instructor: Dr Nguyen Thao Trang School of Biotechnology Semester I 2015-2016 Outlines • Catalytic strategies – – – – Acid-base catalysis Covalent catalysis Metal ion catalysis Approximation catalysis • Regulation – – – – Allosteric Covalent modification Proteolytic cleavage Isozymes Outlines • Catalytic strategies – – – – Acid-base catalysis Covalent catalysis Metal ion catalysis Approximation catalysis • Regulation – – – – Allosteric Covalent modification Proteolytic cleavage Isozymes Acid-base catalysis • Specific functional groups in enzyme structure positioned to: – Donate a proton (act as a general acid), or – Accept a proton (act as a general base) • General acid catalysis: proton transfer from an acid lowers the free energy of a reaction’s transition state • General base catalysis: reaction rate is increased by proton abstraction by a base (a) Uncatalyzed Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt Acid-base catalysis (b) General acid catalyzed (c) General base catalyzed Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt Acid-base catalysis • The side chains of the amino acid residues Asp, Glu, His, Cys, Tyr, and Lys act as acid and/or base catalysts Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt Covalent catalysis • Accelerates reaction rates through the transient formation of a catalyst–substrate covalent bond • Covalent intermediate is more reactive in next step in reaction, so that step has lower activation energy than it would have for a noncovalent catalytic mechanism enzyme alters pathway to get to product • This covalent bond is formed by the reaction of a nucleophilic group on the catalyst with an electrophilic group on the substrate  nucleophilic catalysis Covalent catalysis • Nucleophile: an electron-rich group that attacks nuclei • Electrophile: an electron-deficient group Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt Metal ion catalysis • Nearly 1/3 of all known enzymes require metal ions for catalytic activity • Metal ions can be – Tightly bound (metalloenzymes), i.e., as a prosthetic group (usually transition metal ions, e.g., Fe2+ or Fe3+, Zn2+, Cu2+, Mn2+…) – Loosely bound, binding reversibly and dissociating from enzyme (usually Na+, K+, Mg2+, Ca2+ ) • Functions of metal ions in catalysis: – Binding and orientation of substrate (ionic interactions with negatively charged substrate) – Redox reactions (e.g., Fe2+ / Fe3+ in some enzymes) – Shielding or stabilizing negative charges on substrate or on transition state (electrophilic catalysis) Metal ion catalysis • Example: Carbonic anhydrase H2O polarized by Zn2+ ionizes to form OH which nucleophilically attacks the enzymebound CO2: Active site of carbonic anhydrase Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt 10 Enzyme Regulations • Allosteric regulation: – An allosteric enzyme has two distinct states of its quarternary structure: • • – – T (tense) state (less active): has lower affinity for substrates  lower catalytic activity R (relaxed) state (more active): has higher affinity for substrates  higher catalytic activity At any fixed concentration of substrates, R  T The position of equilibrium depends on the number of active sites that are occupied by substrate Example of ATCase: binding of the inhibitor CTP shifts the equilibrium toward T state (lower affinity to substrate) 74 Biochemistry, Tymoczko, Berge, Strayer Enzyme Regulations • Allosteric regulation: – An allosteric enzyme does not follow Michaelis-Menten kinetics: sigmoidal shape (S) instead of rectangular hyperpolic Sigmoidal curve as binding of substrate to one active site favors the conversion of the entire enzyme to R state  increasing the activity of other active sites  shows “cooperativity” between active sites Sigmoidal curve is a mixture of Michaelis-Menten curves: i) T-state curve with high KM and ii) R-state curve with low KM When [S] increases, equilibrium is shifted from T- to R-state steep rise in activity with respect to [S] 75 Biochemistry, Tymoczko, Berge, Strayer Allosteric regulation • Allosteric regulation: – Allosteric regulators modulate the T- to-R equilibrium CTP inhibition (curve shifted to the right) ATP activation (curve shifted to the left) Decrease in rate ! Increase in rate ! CTP stabilizes the T state making it more difficult for the substrate binding to convert the enzyme to R-state ATP stabilizes the R state making it easier for the substrate binding to convert the enzyme to R-state 76 Biochemistry, Tymoczko, Berge, Strayer Regulation by covalent modification – Covalent attachment of a molecule to an enzyme or protein can modify its activity – Phosphorylation and dephosphorylation are the most common means of covalent modification – Attachment of acetyl groups and their removal are another common means Biochemistry, Tymoczko, Berge, Strayer 77 Regulation by covalent modification • Phosphorylation – Protein phosphorylation is catalysed by protein kinases – More than 550 protein kinases in humans – Terminal phosphoryl group of ATP transferred to Ser or Thr or to a Tyr residue by two main classes of kinases (Serine-/Threoninekinase and Tyrosine-kinase, respectively) – Intracellular protein modification Biochemistry, Tymoczko, Berge, Strayer 78 Regulation by covalent modification • Dephosphorylation – Dephosphorylation of protein is catalyzed by protein phosphotases Biochemistry, Tymoczko, Berge, Strayer – Dephosphorylation is not a reversal of phosphorylation – Both phosphorylation and dephosphorylation are entirely enzyme-dependent (kinetic control) characteristics 79 Regulation by covalent modification • Characteristics of phosphorylation: – Phosphoryl group adds two negative charges leading to structural changes which may affect substrate binding and catalytic activity – Phosphate group allows hydrogen bonding creating a new interaction site – Free energy of phosphorylation is large Part of the energy can thus be used to make conformational changes within the protein favoring a certain state – Phosphorylation and dephosphorylation can be fast (seconds) or slow (hours) Kinetics can be adjusted to physiological requirements – Phosphorylation often induce amplified effects (i.e one kinase molecule can phosphorylate many substrate proteins) – ATP is the cellular energy currency as it couples metabolism to energy status of the cell 80 Enzyme regulation by proteolytic cleavage • Some enzymes are inactive until or a few peptide bonds is cleaved  zymogen or proenzyme • ATP is not required for the proteolytic activation • Cleaved proteins can be intracellular or extracellular • Proteolytic activation takes place once in the life of an enzyme molecule 81 Enzyme regulation by proteolytic cleavage • Proteolytic activation is common for some enzymes and proteins: – Digestive enzymes: synthesized as zymogens in stomach and pancreas – Blood clotting: mediated by a cascade of proteolytic activations – Protein hormones: insulin is derived from proinsulin by cleavage of a peptide – Collagen: derived from procollagen – Programmed cell death, or apoptosis: mediated by proteolytic enzymes called caspases, which are synthesized in precursor form as procaspases 82 Enzyme regulation by proteolytic cleavage • Example: chymotrypsinogen is activated by proteolytic cleavage of a peptide – Chymotrypsin is a digestive enzyme that hydrolyzes proteins in the small intestine, Its inactive precursor, chymotrypsinogen, is synthesized in the pancreas A single polypeptide consisting of 245 amino acids residues Peptide bond joining arginine 15 and isoleucine 16 is cleaved by trypsin -chymotrypsin, then acts on other -chymotrypsin by removing two dipeptides to yield -chymotrypsin Biochemistry, Tymoczko, Berge, Strayer 83 Enzyme regulation by proteolytic cleavage • Example: chymotrypsinogen is activated by proteolytic cleavage of a peptide – Proteolytic activation of chymotrypsinogen leads to the formation of a substrate-binding site - Cleavage of the peptide bond between amino acid 15 and 16 triggers conformational changes: - Alteration of the position of Ile16  electrostatic interaction between Ile16 and Asp 194  triggers conformational changes in the active enzyme: formation of substrate-specificity site Biochemistry, Tymoczko, Berge, Strayer 84 Enzyme regulation by proteolytic cleavage • Generation of trypsin from trisinogen leads to activation of other zymogens – The digestion of proteins in the duodenum requires the concurrent action of several proteolytic enzymes  Trypsin activates these enzymes at the same time – Trypsinogen, the inactivate precursor of trypsin is activated by enteropeptidase  tripsin activates more trypsinogen and other zymogens Biochemistry, Tymoczko, Berge, Strayer 85 Regulation by isozymes • Isozymes = Isoenzymes • Catalyze the same reaction but differ in amino acid sequence • Different kinetic parameters (KM)and regulatory properties • Encoded by different genes and result from duplication of genes • Fine tuning of metabolism in different tissues and developmental stages 86 Regulation by isozymes • Isozymes provides means of regulation specific to different tissues: – Example: Isozymes of lactate dehydrogenase (LDH) – isozyme polypeptide chains: H isozyme and M isozyme – Tetramer in different combinations Biochemistry, Tymoczko, Berge, Strayer Heart: H4 tetramer allosterically inhibited by pyruvate, high substrate affinity; optimal function in aerobic environment Muscle: M4 tetramer not inhibited by pyruvate, low substrate affinity; optimal function in anaerobic 87 environment Regulation by isozymes • Isozymes provides means of regulation specific to developmental stages: – Example: Isozymes of lactate dehydrogenase (LDH) – LDH isozyme profile during development of rat heart: “Anaerobic tissue at early time points, aerobic in the adult animal” M4 H4 Biochemistry, Tymoczko, Berge, Strayer 88 ... rotational motions of their substrates and catalytic groups as in the transition state, reacting groups have little relative motion 11 Examples of catalytic strategies • Chymotrypsin: – A protease,... site Enzyme molecule now in its original state, with His imidazole in neutral form, catalytic triad appropriately hydrogen-bonded, and active site ready to bind another molecule of substrate and. .. Pratt Metal ion catalysis • Nearly 1/3 of all known enzymes require metal ions for catalytic activity • Metal ions can be – Tightly bound (metalloenzymes), i.e., as a prosthetic group (usually transition