Understanding Enzyme Mechanisms and Inhibitors: A Focus on Biochemistry - Prof. Yogarajah , Study notes of Biochemistry

Download Understanding Enzyme Mechanisms and Inhibitors: A Focus on Biochemistry - Prof. Yogarajah and more Study notes Biochemistry in PDF only on Docsity! Biochemistry: A Short Course First Edition Tymoczko • Berg • Stryer © 2010 W. H. Freeman and Company CHAPTER 7 Mechanisms and Inhibitors In this lesson we will investigate how Catalytic strategies of enzymes works and then how enzyme activity can be modulated by environmental factors distinct from allosteric signals Chemical Catalysis   Active site of most enzymes is lined with hydrophobic amino acids.   There are a few polar a.a. which make up the catalytic center of the active site and can be ionized. Histidine (basic a.a.) is common. Aspartate and glutamate - negatively charged Lysine and arginine - positively charged; electrostatic binding can occur Most enzymes commonly employ one or more of the following strategies to catalyze specific reactions 1. Covalent catalysis. Substrate forms a covalent bond with enzymes, then part of substrate is transferred to a second substrate in a 2 step process.   A-X + E X-E + A X-E + B B-X + E The active site contains a reactive group, usually a powerful nucleophile that becomes temporarily covalently modified in the course of catalysis. The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism   2. General acid base catalysis Enzymes that use this have a.a. side chains that can donate or accept electrons to substrate.  Can accelerate a chemical reaction by a factor of 10-100. In general acid –base catalysts, a molecule other than water plays the role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine.   Enzyme activity can be modulated by temperature , pH, and inhibitory molecules Temperature Alters Enzymatic Activity Tyrosinase • part of the pathway that synthesizes the pigment that results in dark fur, has low tolerance for heat. It inactive at normal temperature but functional at lower temperature. • Heat sensitive Tyrosinase Activity Curve Why are Siamese markings only on their extremities? cool enough for tyrosine to gain function and produce pigment Agama – adjust their body temperatures and, hence , the rate of biochemical reactions behaviorally- most active in warmer temperatures- inactive in cooler temperatures. Thermophilic archaea , can live at temperatures of 80 degree Celsius that would denature most proteins. The proteins in these organisms have evolved to be very resistant to thermal denaturation. Competitive Enzyme Inhibition A competitive inhibitor binds at the active site and thus prevents the substrate from binding Reaction Pathway Is Vmax affected? How is KM influenced? Inhibitor resembles the substrate and binds to the active site of the enzyme. Useful drugs – sulfanilamide an antibiotic. Mimics p-aminobenzoic acid (PABA) , a metabolite required by bacteria for the synthesis of folic acid. Read page 94 Ibuprofen – which inhibits a cyclooxygenase that helps to generate the inflammatory response, and statin, which inhibit the key enzyme in chlosterol synthesis. Graphical representation of competitive inhibitors:  affects Km (increases Km --> decreases affinity; need more substrate to reach half- saturation of enzyme) Vmax unaffected Substrate can out compete inhibitor → Vmax unchanged since Vmax= k2[E]As concentration of a competitive inhibitor increases, higher concentrations of substrate are required to attain a particular reaction velocity. The reaction pathway suggests how sufficiently high concentrations of substrate can completely relieve competitive inhibition. Reaction Pathway Competitive Enzyme Inhibition page 95 Reaction PathwaySubstrate can out compete inhibitor → Vmax unchanged since Vmax= k2[E]T Inhibitor binds in the active site → KM increases since KM= (k-1 + k2)/k1 e.g. AZT inhibition of HIV reverse transcriptaseactual substrate is dTTP deoxythymidine triphosphate) Noncompetitive Enzyme Inhibition Reaction Pathway Taking enzyme out of circulation → Vmax lowered since Vmax= k2[E] Inhibitor binds both E and ES → KM unchanged since KM= (k-1 + k2)/k1 Noncompetitive Inhibition -—Lineweaver-Burk Plot ° V wax Lowered ¢ K,, Unchanged 1/Vo| Slope = Ky/Vinax Intercept = -1/Kjy Intercept = 1/V nax ° 1/[S] + Noncompetitive f/ inhibitor XN No inhibitor present WV 0 1/[S] Uncompetitive Enzyme Inhibition Inhibitor binds only to the enzyme –substrate complex. Reaction Pathway Uncompetitive Inhibition - Lineweaver-Burk Plot + Unncompetitive inhibitor —” ° Va, Lowered <_< No inhibitor ¢ K,, Lowered present 1/V 0 1/[S] Inhibitor Identification • No Inhibition? • Competitive Inhibition? • Uncompetitive Inhibition? • Noncompetitive Inhibition? no n un com Chymotrypsin Specificity- shows basic principles of catalysis and inhibition Cleaves proteins on the carboxyl side of aromatic or large hydrophobic amino acids ( shaded) – the red bonds indicates where enzyme acts. Active Site Mapping via Irreversible Inhibitors Diisopropylphosphofluoridate (DIPF) inhibits chymotrypsin by modifying 1 of 28 serine residues Affinity labels also called substrate analogs Structurally similar to the substrate More specific for an enzymes' active site than are group specific reagents. Active Site Mapping via Irreversible Inhibitors Affinity labels Modifies an essential histidine residue Cell Wall Cross-Linking in Staphylococcus aureus The formation of cross links in bacterial peptidoglycan. The terminal amino group of the pentaglycine bridge in the cell wall attacks the peptide bond between two D-alanine residues to form a cross link. Penicillin mimics a normal substrate to enter the active site. To create cross links between the tetrapeptide and pentaglycines, the tetrapeptides normally forms an acyl intermediate with the penultimate D- alanine residue of the tetrapeptide . This covalent acyl-enzyme intermediate then reacts with the amino group of the terminal glycine in another peptide to form the cross link. Penicillin mimics a normal substrate to enter the active site. Penicillin is welcomed into the active site of the transpeptidase because it mimics the D -Ala-D-Ala moiety of the normal substrate. On binding to the transpeptidase , the serine residue at the active site attacks the carbonyl carbon atom of the lactam ring to form the penicilloyl –serine derivative. This penicilloyl –serine derivative does not react further-cell wall synthesis stop – penicillin acts as a sucide inhibitor. All three enzymes have similar primary, second, tertiary structure.  All cleave peptide bonds on COOH side of hydrophobic or aromatic side chains.  Substrate specificity is due to amino acid residues in the hydrophobic binding pocket. chymotrypsin - serine (uncharged) --> accepts large, bulky, hydrophobic side groups.   trypsin - aspartate ( negatively charged) accepts Lys, Arg, Gly, Ala. elastase - shallow - binds a.a. with small side chains ( Gly, Ala).   Chymotrypsin employs to attack the substrate carbonyl group. Chymotrypsin contains an extra ordinarily reactive serine residue. Chymotrypsin Catalysis Proceeds via a Two-Step Mechanism Product –p-nitrophenolate –measurements of the absorbance of light revealed the amount of p-nitrophenolate being produced and thus provided a facile means of chymotrypsin activity. Under steady state conditions, -substrate obeys michael-menton kinetics. Initial rapid burst of color product, followed by its slower formation. This results suggests that hydrolysis proceeds in two steps. 1) Substrate enters enzyme and is aligned with R1 group in binding pocket --> places carbonyl carbon of peptide bond next to oxygen of Ser- 195. Chymotrypsin Catalytic Triad •1. Catalytic triad serves as the site of catalysis-A catalytic triad refers to the three amino acid residues found inside the active site of certain protease enzymes: serine, aspartate , and histidine . They work together to break peptide bonds on polypeptides. •2.Aspartate and histidine contribute serine’s basicity • 3.Serine serves as a nucleophile in covalent catalysis- A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction 2) His-57 attacks H of Ser-195. 3) Now, the nucleophilic oxygen of Ser-195 attacks carbonyl carbon of peptide bond to form tetrahedral intermediate (transition state?). Substrate binding via nucleophilic attack 6) His-57 imidazolium ring acts as an acid catalyst by donating H to peptide bond --> molecule cleaved --> amine product released. Polypeptide original N-side serves as leaving group and enzyme is regenerated 7) Carbonyl group of peptide forms covalent bond with enzyme --> acyl-enzyme intermediate formed. Polypeptide original N-side serves as leaving group and enzyme is regenerated 8) After first product leaves, a molecule of water enters --> donates H+ to His-57 --> -OH group left attacks carbonyl group --> formation of second tetrahedral intermediate and stabilized by oxyanion hole plus a low barrier H-bond.Polypeptide original N-side serves as leaving group and enzyme is regenerated