Pharmaceutical Biochemistry LEC Past Paper

CYCLE SE Pharmaceutical Biochemistry LEC PBSCI3 | BERNARDIO Holoenzyme – biochemically active conjugated MODULE 4: ENZYMES enzyme produced from an apoenzyme and a cofactor...

CYCLE SE Pharmaceutical Biochemistry LEC PBSCI3 | BERNARDIO Holoenzyme – biochemically active conjugated MODULE 4: ENZYMES enzyme produced from an apoenzyme and a cofactor WHAT ARE ENZYMES Apoenzyme + Cofactor = Holoenzyme Enzymes a compound, usually a protein, that acts as a Coenzyme – small organic molecule that serves as a catalyst for a biochemical reaction. cofactor in a conjugated enzyme Most enzymes are globular proteins COFACTOR Enzymes undergo all the reactions of proteins Common coenzymes are vitamins and metal ions including denaturation Metal ion activators are inorganic and may be Enzymes cause cellular reactions to occur bonded through coordinate covalent bonds millions of times faster than corresponding Typical inorganic ion cofactors: uncatalyzed reactions. 1) Zn 2) Mg 3) Mn 4) Fe 5) Cu 6) K 7) Na Enzyme activity is dramatically affected by: (a) temperature (b) alterations in pH (c) other protein denaturants Cofactor Enzyme Enzyme Structure Coenzyme Two General Structural Classes: Thiamine pyrophosphate Pyruvate a) Simple Enzyme – composed only of protein dehydrogenase (amino acid chains) Flavin adenine nucleotide Monoamine oxidase b) Conjugated Enzyme – has a nonprotein part in Nicotinamide adenide addition to a protein part Lactate dehydrogenase nucleotide o Apoenzyme – protein part of the conjugated Pyridoxal phosphate Glycogen enzyme phosphorylase Coenzyme A (CoA) Acetyl CoA carboxylase ▪ the inactive form of apoenzyme is known as Proenzyme / Zymogen Biotin Pyruvate carboxylase o Cofactor (activator) – also known as coenzymes 5’ – Deoxyadenosyl Methylmalonyl mutase cobalmin ▪ nonprotein part of a conjugated Tetrahydrofolate Thymidylate synthase enzyme *able to provide additional chemically reactive functional groups AD | PHARMACY 2A | 2021 1  MODULE 4: ENZYMES 2) The type of reaction catalyzed by an enzyme is Metal often noted with a prefix. An oxidase enzyme catalyzes an oxidation reaction, and a hydrolase Zn2+ Carbonic anyhydrase enzyme catalyzes a hydrolysis reaction. Zn2+ Carboxypeptidase 3) Identity of substrate is often used in addition to the Mg2+ EcoRV type of reaction. Mg2+ Hexokinase E.g. Glucose oxidase, pyruvate carboxylase, and Ni2+ Urease succinate dehydrogenase Mo Nitrate reductase Six Major Classes of Enzymes Based on the Se Glutathione peroxidase Types of Reactions they Catalyze Mn2+ Superoxide dismutase Oxidoreductase K+ Propionyl CoA is an enzyme that catalyzes an oxidation carboxylase reduction reaction requires a coenzyme that is oxidized or reduced Vitamin Coenzyme Function as the substrate is reduced or oxidized Example: Lactate Dehydrogenase oxidation or nicotinamide adenine Transferase niacin hydrogen dinucleotide (NAD+) an enzyme that catalyzes the transfer of a transfer oxidation or functional group from one molecule to another flavin adenine riboflavin hydrogen A transaminase catalyzes the transfer of an dinucleotide (FAD) transfer amino group from one molecule to another. pantothenic acetyl group Kinases, which play a major role in metabolic coenzyme A (CoA) acid carrier energy-production reactions, catalyze the methyl transfer of a phosphate group from adenosine vitamin B-12 coenzyme B-12 group transfer triphosphate (ATP) to give adenosine aldehyde diphosphate (ADP) and a phosphorylated thiaminpyrophosphate product thiamin (B-1) group (TPP) Hydrolase transfer an enzyme that catalyzes a hydrolysis reaction Prosthetic Groups in which the addition of a water molecule to a are tightly incorporated into protein structure by bond causes the bond to break covalent or noncovalent forces Carbohydrases effect the breaking of examples include derivatives of B vitamins such glycosidic bonds in oligo- and polysaccharides, as pyridoxal phosphate, flavin mononucleotide proteases effect the breaking of peptide (FMN), flavin adenine dinucleotide (FAD), linkages in proteins, and lipases effect the thiamin pyrophosphate, biotin, and metal ions of breaking of ester linkages in triacyglycerols Co, Cu, Mg, Mn, and Zn. Lyase an enzyme that catalyzes the addition of a Nomenclature of Enzymes group to a double bond or the removal of a 1) The suffix -ase identifies a substance as an group to form a double bond in a manner that enzyme. Thus urease, sucrase, and lipase are all does not involve hydrolysis or oxidation enzyme designations. A dehydratase effects the removal of the components of water from a double bond and a The suffix -in is still found in the names of some of hydratase effects the addition of the the first enzymes studied, many of which are compositions of water to a double bond. digestive enzymes. Such names include trypsin, chymotrypsin, and pepsin. AD | PHARMACY 2A | 2021 2  MODULE 4: ENZYMES Isomerase an enzyme that catalyzes the isomerization (rearrangement of atoms) of a substrate in a reaction, converting it into a molecule isomeric with itself Ligase an enzyme that catalyzes the bonding together of two molecules into one with the participation of ATP CLASS TYPE OF REACTION EXAMPLE Oxidoreductase Oxidation-reduction Lactate dehydrogenase Nuceloside monophosphate Transferases Group transfer kinase (NMP kinase) Hydrolases Hydrolysis reactions (transfer of functional groups to water) Chymotrypsin Lyases Addition or removal of groups to form double bonds Fumarase Isomerases Isomerization (intramolecular group transfer) Triose phosphate isomerase Ligases Ligation of two substrates at the expense of ATP hydrolysis Aminoacyl-tRNA synthase Models of Enzyme Action LOCK-AND-KEY MODEL Active Site relatively small part of an enzyme’s structure that the active site in the enzyme has a fixed, rigid is actually involved in catalysis geometrical conformation. Only substrates with a complementary geometry can be Enzyme – Substrate Complex accommodated at such a site, much as a lock accepts only certain keys intermediate reaction species that is formed when a substrate binds to the active site of an enzyme INDUCED - FIT MODEL allows for small changes in the shape or geometry of the active site of an enzyme to accommodate a substrate. A good analogy is the changes that occur in the shape of a glove when a hand is inserted into it. The induced fit is a result of the enzyme’s flexibility; it adapts to accept the incoming substrate AD | PHARMACY 2A | 2021 3  MODULE 4: ENZYMES STEREOCHEMICAL SPECIFICITY enzyme will act on a particular stereoisomer. Chirality is inherent in an enzyme active site because amino acids are chiral compounds An L-amino acid oxidase will catalyze the oxidation of the L-form of an amino acid but not the D-form of the same amino acid Factors that Affect Enzyme Activity Enzyme Specificity Enzyme Activity extent to which an enzyme’s activity is restricted a measure of the rate which an to a specific substrate, a specific group of enzyme converts substrate to substrate, a specific type of chemical bond or a products in a biochemical specific type of reaction reaction The degree of enzyme specificity is determined four factors: o temperature by the active site. o pH o substrate concentration ABSOLUTE SPECIFICITY o enzyme concentration enzyme will catalyze only one reaction. This TEMPERATURE most restrictive of all specificities is not common When the temperature increases beyond a Catalase is an enzyme with absolute certain point, the increased energy begins to specificity. It catalyzes the conversion of cause disruptions in the tertiary structure of the hydrogen peroxide (H2O2) to O2 and H2O. enzyme; denaturation is occurring Hydrogen peroxide is the only substrate it will Change in tertiary structure at the active site accept. impedes catalytic action, and the enzyme activity quickly decreases as the temperature climbs past this point GROUP SPECIFICITY Optimum temperature (7 – 7.5) is the temperature at which an enzyme exhibits maximum activity the enzyme will act only on molecules that have a specific functional group, such as hydroxyl, amino, or phosphate group PH Carboxypeptidase is group-specific; it cleaves small changes in pH (less than one unit) can amino acids, one at a time, from the carboxyl result in enzyme denaturation and subsequent end of a peptide chain loss of catalytic activity most enzymes exhibit maximum activity over a LINKAGE SPECIFICITY very narrow pH range Optimum pH is the pH at which an enzyme enzyme will act on a particular type of chemical exhibits maximum activity bond, irrespective of the rest of the molecular structure SUBSTRATE CONCENTRATION Phosphatases hydrolyze phosphate-ester As substrate concentration increases, the point bonds in all types of phosphate esters. Linkage is eventually reached where enzyme specificity is the most general of the common capabilities are used to their maximum event. specificities. Each substrate must occupy an enzyme active site for a finite amount of time, and the products must leave the site before the cycle can be AD | PHARMACY 2A | 2021 4  MODULE 4: ENZYMES repeated. When each enzyme molecule is working at full capacity, the incoming substrate molecules must “wait their turn” for an empty active site. Turnover Number is the number of substrate molecules transformed per minute by one molecule of enzyme under optimum conditions of temperature, pH, and saturation NONCOMPETITIVE INHIBITOR ENZYME CONCENTRATION a molecule that decreases enzyme activity by binding to a site on an enzyme other than the If the amount of substrate present is kept constant active site and the enzyme concentration is increased, the reaction rate increases because more substrate molecules can be accommodated in a given amount of time -increase enzyme concentration - increase rate of reaction IRREVERSIBLE INHIBITOR Enzyme Inhibition Enzyme Inhibitor – slows or stops the normal catalytic a molecule that inactivates enzymes by forming a function of an enzyme by binding to it strong covalent bond to an amino acid sidechain group at the enzyme’s active site COMPETITIVE INHIBITOR a molecule that sufficiently resembles an enzyme substrate in shape and charge distribution that it can compete with the substrate for occupancy of the enzyme’s active site AD | PHARMACY 2A | 2021 5  MODULE 4: ENZYMES Enzymes in Clinical Use DRUGS THAT INHIBIT ENZYME ACTIVITY certain targeted enzymes 1. Statins – HMG Coenzyme A reductase inhibitors (3- hydroxy-3-methylgutaryl); lower serum lipid concentration o reduce cholesterol synthesis 2. Emtrictabine and Tenofovir Disoproxil Fumarate – inhibitors of viral reverse transcriptase; block replication of HIV o Angiotensin Converting Enzyme (ACE) o inhibit the ability to convert/create Angiotensin II ® narrow blood vessels ® higher blood pressure o main mechanism: interfere with body’s RADS (Renin Angiotensin Aldosterone System) 3. ACE Inhibitors (Captopril, Lisinopril, Enalapril) – antihypertensive agents 4. Lactam Antibiotics (Penicillin and Amoxicillin) – inhibitors of alanyl alanine carboxypeptidasetranspeptidase, thus blocking cell wall synthesis o beta lactum urine in their structure 5. Sulfa Drugs – mimic PABA ® no growth of microorganisms o Para Amino Benzoic Acid ▪ vital importance for folic synthesis ▪ bacterial enzyme systems SELECTED BLOOD ENZYME ASSAYS USED IN DIAGNOSTIC MEDICINE possible tissue damage / leakage of cellular contents in the bloodstream heart disease liver Lactate Dehydrogenase (LDH) disease Creatinine Phosphokinase (CPK) heart disease heart disease liver Aspartate Transaminase (AST) disease AD | PHARMACY 2A | 2021 6  MODULE 4: ENZYMES muscle damage Water-Soluble Vitamins Fat-Soluble Vitamins heart disease Vitamin C Vitamin A Alanine Transaminase (ALT) liver disease muscle damage Thiamin Vitamin D Gamma-glutamyl Transpeptidase heart disease liver (GGTP) disease Riboflavin Vitamin E bone disease liver Niacin Vitamin K Alkaline Phosphatase (ALP) disease GENERAL CHARACTERISTICS OF Pantothenic acid VITAMINS Vitamin B6 Vitamins is an organic compound, essential in small Biotin amounts for the proper functioning of the human body, that must be obtained from dietary sources because the body Folate cannot synthesize it. Vitamin B12 Water-Soluble Vitamins (B Fat-Soluble Vitamins (Vitamins Vitamins and Vitamin C) ADEK) absorption directly into the blood first enter into the lymph system transport travel without carriers many require protein carriers circulate in the water-filled parts of the storage body found in the cells associated with fat excretion kidneys remove excess in urine tend to remain in fat-storage sites not likely to reach toxic levels when likely to reach toxic levels when toxicity consumed from supplements consumed from supplements dosage frequency needed in frequent doses needed in periodic doses relationship to coenzymes function as coenzymes do not function as coenzymes Vitamin B The preferred and alternative names for the B vitamins: Folate – folic acid Thiamin – Vitamin B1 Vitamin B12 – cobalmin Riboflavin – Vitamin B2 Pantothenic acid – Vitamin B5 Niacin – Nicotinic acid, Nicotinamide, Vitamin B3 Biotin – Vitamin B7 Vitamin B6 – Pyridoxine, Pyridoxal, Pyridoxamine *Exhibit structural diversity *Major function: B Vitamins are components of coenzymes B VITAMIN COENZYMES GROUPS TRANSFERRED thiamin thiamin pyrophosphate (TPP) aldehydes flavin mononucleotide (FMN) flavin riboflavin adenine dinucleotide (FAD) hydrogen atoms nicotinamide adenine niacin dinucleotide (NAD+) nicotinamide hydrogen atoms adenine AD | PHARMACY 2A | 2021 7  MODULE 4: ENZYMES dinucleotide phosphate (NADP+) pantothenic acid coenzyme A (CoA) acyl groups pyridoxal-5-phosphate (PLP) pyridoxine-5’- vitamin B6 phophate (PNP) pyridoxamine-5’-phosphate amino groups (PMP) biotin biotin carbon dioxide (Carboxyl group) folate tetrahydrofolate (THF) one-carbon groups other than CO2 vitamin B12 methylcobalamin methyl groups , hydrogen atoms Vitamin A Vitamin E Four Major Functions: 1. Vision Alpha-tocopherol is the most active biological 2. Regulating Cell Differentiation active form of Vitamin E 3. Maintenance of the Health of Epithelial Tissue o Myra-E Supplement 4. Reproduction and Groups Peanut oils, green and leafy vegetables and whole grain products are the sources of vitamin Vision: In the eye (0.1%) – vitamin A combines with E opsin protein to form the visual pigment rhodopsin Primary function: Antioxidant – protect against which further converts light energy into nerve impulses oxidation of other compounds that are sent to the brain o prevent oxidation of PUFAs o protect vit A from undergoing oxidation Regulating Cell Differentiation – process in which immature cells change to specialized cells with function Vitamin K Maintenance of the health of epithelial tissues via Two major forms: K1 and K2 o K1 is epithelial tissue differentiation – lack of vitamin A found in dark green, leafy vegetables causes such surfaces to become drier and harder than o K2 is synthesized by bacteria that grow in normal colon Dietary need supply: 1/2 synthesized by Reproduction and Growth – In men, vitamin A bacteria and 1/2 obtained from diet participates in sperm development. In women, normal Active in the formation of proteins fetal development during pregnancy requires vitamin A (prothrombin) involved in regulating blood e.g. immature bone marrow cells ® undergo cell clotting differentiation = WBC / RBC given to patients who undergo surgery ® prevent hemorrhage Vitamin D Two forms active in the body: Vitamin D2 and D3 The two most important members of the vitamin D family of molecules are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) Vitamin D3 is produced in the skin of the humans and animals by the action of sunlight (ultraviolet light) on its precursor molecule, the cholesterol derivative 7-dehydrocholesterol (a normal metabolite of cholesterol found in the skin) AD | PHARMACY 2A | 2021 8