5. Intro to Enzymes Flashcards
6 of The Top 10 Prescribed Drugs Target Enzymes
Acetaminophen –cyclooxygenase 1 and 2
Simvastatin (Zocor) – HMG CoA reductase
Lisinopril – Angiotensin-converting enzyme (ACE)
Prilosec – H+/K+ ATPase (proton pump)
Azithromycin (Z-pack) – Peptidyl transferase (bacterial 50s ribosome)
Amoxicillin – Transpeptidase (bacterial cell wall synthesis)
Enzyme
Biological catalyst that speeds up chemical transformation of biological molecules
Accelerates the rate of a reaction but is usually not altered itself in the process.
Most enzymes are proteins
- not all proteins function as enzymes
- Some non-proteins may function as catalysts (e.g., ribozymes)
Substrate(s) and Product(s)
Substrate: molecule being transformed by the enzyme.
Product: result of an enzymatic transformation.
Cofactors and Coenzymes
Non-protein organic or inorganic molecules that assist an enzyme in the catalytic process
Cofactors: metal ions that associate with an enzyme via non-covalent interactions
Coenzymes: organic molecules (mostly derived from vitamins) that associate with enzymes, either loosely (diffusible, cosubstrate) or tightly (non-diffusible, prosthetic group)
Holoenzyme: Enzyme + cofactor/coenzyme
Apoenzyme : Holoenzyme – cofactor/coenzyme
Advantages of Enzymes
increase reaction rates by many-fold (typically 109-fold)
enable reactions under mild conditions (37°C, pH 7.0)
increase reaction specificity
enable regulation
eg pyrrolyl-3-acetate -> 3 methylpyrrole
Uncatalyzed half-life = 2.9 x 109 years at 25°C
Catalyzed rate = 10 mol per min
Rate enhancement = 1.2 × 1017
Enzymes do not affect Keq i.e. [Product]/[Substrate]
Active Site of an Enzyme
a three dimensional pocket or cleft that binds to the substrate (a.k.a. catalytic site)
where the conversion of a substrate to a product takes place
constituted by amino acids that may be in different regions of the enzyme protein
the result of folding in the tertiary structure of the protein
Enzyme Specificity
In the above example, the active site binds to the hydroxyl group, methyl group and one of the two hydrogens
The stereospecificity of the active site allows it to distinguish between the two hydrogens
How Enzymes Accelerate a Reaction
They lower the free energy of activation (E of transition state minus E of reactants) to make a substrate reach the transition state easier
Enzymes do not alter
- ΔG (free E of overall rxn [E of reactants minus E of products]), or
- Equilibrium constant
Enzymes are Classified by Reaction Type
- oxidoreductases- catalyze oxidation-reduction rxns eg lactacte -> pyruvate
- transferases- cat transfer of C-, N-, or P-containing gps eg serine -> glycine
- hydrolases- cat cleavage of bonds by addn of water, eg urea + H2O -> CO2 + 2NH3
- lyases- cat cleavage of C-C, C-S, certain C-N bonds eg pyruvate -> acetaldehyde
- isomerases- cat racemization of optical or geometric isomers, eg methylmalonyl CoA -> succinyl CoA
- ligases- cat formation of bonds btwn carbon and O, S, N coupled to hydrolysis of high-E phosphates eg pyruvate -> oxaloacetate
- Oxidoreductases
Transfer electrons from donor (reducing agent) to acceptor (oxidizing agent) Dehydrogenases SH2 → S Oxidases O2 → H2O2 Reductases NADH + Sox → Sred + NAD+ Peroxidases H2O2 + Sred → Sox + H20 Catalase H2O2 → O2 + H2O Oxygenases S + O2 → SO + H2O Hydroxylases S + O2 → SOH + H2O
- Transferases
Transfer functional gp (eg amino, phosphate) btwn molecules Transaminases Phosphotransferases Kinases (High energy phosphate) Methylases Sulfotransferases Phosphomutases Phosphorylases
4,5. Isomerases and Lyases
Racemases: a subclass of isomerases (rearrange/isomerize molecs) that interconvert stereoisomers such as D- and L-forms
C-C-Lyases (lyases=synthases- add or remove atoms eg elements of water, ammonia, CO2, to or from double bond) : break a carbon-carbon bond without using water (e.g., HMG-CoA lyase)
- An example of a lyase that adds or removes an entire molecule of water is Fumarase
- Lyases make new bonds (synthases) without directly using the energy of ATP
3,6. Ligases and Hydrolases
Synthetases or ligases form new covalent bonds (C-O, C-S, C-N, C-C) using the energy of hydrolysis of ATP (Unlike synthases )
Hydrolases (cleave bonds via addn of water): glycosidases, proteases, lipases, nucleases and phosphatases
Analytes in Biological Fluids
Immobilized enzymes are used to measure the levels of metabolites in biological samples such as whole blood, serum or urine
- Blood glucose (glucose oxidase)
- Plasma triglycerides (lipase)
- Plasma cholesterol (cholesterol oxidase)
Enzyme-linked immunosorbent assays (ELISA) detect the presence of specific antigens
- Early pregnancy tests (hCG)
- Infections (e.g., HIV)
- In sandwich ELISA, horse radish peroxidase-linked second antibody is used to detect the presence of primary antibody
Principle of Sandwich ELISA
Target protein in a sample is captured by an immobilized primary antibody
A secondary antibody tagged with biotin then reacts with target protein
Biotin is bound by streptavidin linked to horse radish peroxidase (HRP)
