Enzymes And Vitamins Biochemistry PDF

BIOCHEMISTRY BS in Nursing Enzymes and Vitamins 1st Semester Enzymes - In 1897, Eduard Buchner demonstrated cell-free yeast extract...

BIOCHEMISTRY BS in Nursing Enzymes and Vitamins 1st Semester Enzymes - In 1897, Eduard Buchner demonstrated cell-free yeast extract could ferment sugar to alcohol. He also showed fermentation was promoted by molecules that continued to function when removed from cells - The name enzymes were given to the molecules detected by Buchner. - Enzymes are polymers which catalyze biochemical reactions. Most of them are proteins. Like all catalysts, they are neither consumed nor permanently altered after the reaction. Prosthetic group General functions of enzymes: − cofactor/coenzyme covalently bonded Breakdown of nutrients to supply energy to the enzyme and building blocks Assembly of building blocks into proteins, Holoenzyme DNA, membranes, cells, and tissues − complete, catalytically active enzyme Harnessing of energy to power cell mobility together with its bound coenzyme and muscle contraction and/or metal ions Structure Apoenzyme/ apoprotein - Enzyme are protein except for ribozymes − a protein portion without a cofactor, (RNA molecules with catalytic activity) does not have enzymatic activity - Catalytic activity depends on the integrity − inactivite enzyme of their native protein conformation - Primary, secondary, tertiary, and quaternary 3D structure of enzyme structure of protein enzyme are essential to their catalytic activity very large multi- enzyme complex (E1, E2, and E3) Cofactor breaking down − inorganic ions of branched- − Examples: (Fe3+, Mg2+, Mn2+, and chain amino Zn2+) acids Val, Leu, and Ile in foods specific activities of these enzymes and the intricate interplay between the enzyme in the complex ensures that these amino acid in the human diet are properly metabolized in a series of enzymatic steps Example of E1 enzyme has 3 types of protein chains: alpha and beta 4 subunits (tetramer, α2β2) Active sites are within the enzyme Coenzyme: Thiamin diphosphate Coenzymes (Vitamin B1) − more complex organic molecules; Cofactor: K+ derived from vitamins precursors BSN 1A – BIOCHEM: ENZYMES AND VITAMINS  Tyrosine residue substituted by an Induce Fit Model (Daniel E. Koshland, Jr., Asparagine residue at position 393 1958) (Y393N) − The enzyme changes shape on − This class mutations may prevent or substrate binding. The active site forms cause an improper and unstable a shape complementary to the substrate assembly of the subunits into the after the substrate has been bound. tetrameric enzyme. − Maple Syrup Urine Disease Function uncatalyzed reaction – slow catalyzed reaction – fast Active site - a region on an enzyme that binds to a protein or other substance during a reaction. Types of Enzyme Specificity Substrate – a molecule that is bound in the 1. ABSOLUTE SPECIFICITY active site and is acted upon by the enzyme. - enzyme that catalyze a single reaction undergone by a single substance. Example: glucokinase – catalyze the Where; phosphorylation of glucose E–enzyme, S –substrate, P –product ES –enzyme substrate complex 2. GROUP SPECIFICITY EP –enzyme product complex - enzyme that catalyze a single reaction undergone by a group substance that are Catalyst increase the rate of the reaction chemically related or with the chemical Catalyst do not affect the reaction equilibria properties. Example: o alcohol dehydrogenases – catalyze Enzyme specificity dehydrogenation of alcohols − The ability to discriminate between a o hexokinases – catalyze phosphorylation of substrate and a competing molecule. 6-carbon monosaccharides − Derived from the formation of many weak interaction between the enzyme and 3. STEREOCHEMICAL SPECIFICITY specific substrate. - type of enzyme that catalyze a reaction undergone by specific isomers. Example: Only a substrate with –OH group will Example: bind with the specific Glu residue on the o D-Lactate dehydrogenase – catalyze the enzyme, any molecule lacking a –OH group is a breakdown of D-sugars but not L-sugars. poor substrate for the enzyme. 4. LINKAGE SPECIFICITY Lock and Key Model (Emil Fischer, 1894) - type of enzyme that catalyze the cleavage of − The active site of the unbound enzyme specific linkages. is complementary in shape to the Example: substrate. o Lactase – catalyze cleavage of β 1,4 linkages between glucose and galactose o Sucrase – catalyze cleavage of α1- β2linkages between glucose and fructose BSN 1A – BIOCHEM: ENZYMES AND VITAMINS  Factors Affecting Enzyme Activity 4. Concentration of Enzyme 1. Temperature - When the - At 0°C and 100°C, concentration of the the rate of enzyme- enzyme is catalyzed reactions significantly lower is nearly zero. than the concentration of the substrate, the rate of an enzyme-catalyzed reaction is directly dependent on the 2. pH (Hydrogen Ion Concentration) enzyme concentration. This is true for any - Enzymes may be denatured by extreme catalyst; the reaction rate increases as the levels of hydrogen ions (whether high or concentration of the catalyst is increased. low); any change in pH, even a small one, alters the degree of ionization of an Enzyme Inhibition enzyme’s acidic and basic side groups and the substrate components as well. - Ionizable side groups located in the active site must have a certain charge for the enzyme to bind its substrate. Neutralization of even one of these charges alters an enzyme’s catalytic activity. - An enzyme exhibits maximum activity over the narrow pH Inhibitors range in which a - Substance that reduce the activity of an molecule exists in enzyme-catalyzed reaction its properly charged - Act by either directly or indirectly form. The median influencing the catalytic properties of the value of this pH active site range is called the optimum pH of the - Inhibitors can be foreign to the cell or enzyme natural components of it which represent an important element of the regulation of cell 3. Concentration of Substrate metabolism. - In the presence of a - Many toxins and also many given amount of pharmacologically active agents act by enzyme, the rate of an inhibiting specific enzyme-catalyzed enzymatic reaction processes. increases as the substrate concentration Reversible inhibitors increases until a - inhibitor binds reversibly to an enzyme limiting rate is reached, after which further increase in the substrate concentration 1. Competitive inhibition produces no significant change in the - Occurs when substrate (S) and inhibitor (I) reaction rate. both bind to the same site on the enzyme. In - At this point, so much substrate is present effect, they compete for the active site and that essentially all of the enzyme active sites bind in a mutually exclusive fashion. have substrate bound to them. In other - The inhibitor is a molecule that is words, the enzyme molecules are saturated structurally similar to the normal substrate with substrate. and can bind to the enzyme’s active site - The excess substrate molecules cannot react until the substrate already bound to the enzymes has reacted and been released (or been released without reacting). BSN 1A – BIOCHEM: ENZYMES AND VITAMINS  Regulation of enzyme activity Enzymes can be regulated by changing the activity of a preexisting enzyme or changing the amount of an enzyme. A. Changing the activity of a pre-existing enzyme 1. Substrate availability - Substrates (reactants) bind to enzymes with a characteristic affinity, characterized by a 2. Non-competitive inhibition kinetic parameter (Km). If the actual - Occurs when the inhibitor (I) binds to the concentration of a substrate in a cell is much enzyme at a site that is distant from that of less than the Km, the activity of the enzyme the substrate is very low. If the substrate concentration is - In effect, the noncompetitive inhibitor alters much greater than Km, the enzyme active the conformation of the enzyme, such that it site is saturated with substrate and the has reduced or limited function as a catalyst enzyme is maximally active. - The binding of the inhibitor does not physically block the substrate binding site 2. Product inhibition - Inhibitors are chemically unrelated to the - A product of an enzyme-catalyzed reaction substrate often resembles a starting reactant, so it should be clear that the product should also bind to the active site, albeit probably with lower affinity (competitive inhibitor). Under conditions in which the product of a reaction is present in high concentration, it would be energetically advantageous to the cell if no more product was synthesized. Product inhibition is hence commonly observed. Likewise it be energetically advantageous to a cell if the end product of an entire pathway could likewise bind to the initial enzyme in the pathways and inhibit 3. Uncompetitive inhibition it, allowing the whole pathway to be - Occurs when the inhibitor is only able to inhibited. This type of feedback inhibition bind to the enzyme once a substrate is commonly observed. molecule has itself bound - Most significant at high substrate 3. Allosteric regulation concentrations, and results in a reduction in - Molecules that bind to sites on target the Vmax of the reaction enzymes other than the active site (allosteric - Binding of the inhibitor to the ES complex sites) can regulate the activity of the target effectively removes ES complex and enzyme (in the same way as noncompetitive thereby affects the overall equilibrium of inhibitor). These molecules can be the reaction favoring ES complex formation structurally dissimilar to those that bind at the active site. They do so my conformational changes which can either activate or inhibit the target enzyme's activity. One type of very common allosteric regulation is feedback inhibition. In this type of allosteric regulation, the end product of a metabolic pathways inhibits the first enzyme in the pathway by binding to an allosteric site. BSN 1A – BIOCHEM: ENZYMES AND VITAMINS  Feedback inhibition vs. Product inhibition decreasing translation of the protein enzyme from its mRNA. 3. Post-translational changes - Once a protein enzymes is translated from 4. Covalent modification its mRNA, it can undergo a changes to - Many if not most proteins are subjected to affect enzyme levels. Some proteins are post-translational modifications which can synthesized in a "pre-form which must be affect enzyme activity through local or cleaved in a targeted and limited fashion by global shape changes, by promoting or proteases to active the protein enzyme inhibiting binding interaction of substrates (proteolytic activation). This type of and allosteric regulators, and even by enzymes are called zymogens. The changing the location of the protein within precursor is often called a proenyzme. the cell. Proteins may be phosphorylated, Examples of zymogens are enzymes acetylated, methylated, sulfated, involved in protein degradation (proteases) glycosylated, hydroxylated,etc., often in a and programmed cell death (apoptosis). reversible fashion. Some of these Some proteins are not fully folded and must modifications are reversible. Regulation by bind to other factors in the cell to adopt a phosphorylation through the action of catalytically active form. Finally, fully kinases, and dephosphorylation by active protein can be fully proteolyzed by phosphates is extremely common. Control the proteasome, a complex within cells, or of phosphorylation state is mediated in lysosomes, which are organelles within through signal transduction process starting cells containing proteolytic enzymes. at the cell membrane, leading to the activation or inhibition of protein kinases and phosphatases within the cell. B. Changing the amount of enzyme (genetic control) Another and less immediate but longer duration method to modulate the activity of an enzyme is to alter the activity of an enzyme that already exists in the cell. The list below, shows way in which enzyme concentration is regulated. 1. Alternation in the transcription of enzyme's gene - Extracellular signal (hormones, neurotransmitters, etc) can lead to signal transductions responses and ultimate activation or inhibition of the transcription of the gene for a protein enzyme. These changes result from recruitment of transcription factors (proteins) to DNA Antibiotics that inhibit enzyme activity sequences that regulate transcription of the enzyme gene. Antibiotics - are medication used to fight bacterial 2. Degradation of messenger RNA for the infections enzyme - Originally the term antibiotics referred to - The levels of messenger RNA for a protein natural compound produced by certain will directly determine the amount of that microorganisms for the purpose of fending protein synthesized. Small inhibitor RNAs, off others. derived from microRNA molecules - For example, penicillin: produced by the transcribed from cellular DNA, can bind to fungus specific sequences in the mRNA of a target enzyme. The resulting double-stranded RNA complex recruits an enzyme (Dicer) that cleaves the complex with the effect of BSN 1A – BIOCHEM: ENZYMES AND VITAMINS Mechanism of Action: Cell wall Sites of action of antibiotics that interfere - Bacterial cells surrounded by cell walls with bacterial protein synthesis. made of peptidoglycan - Antibiotics that affect cell wall act as different stages of peptidoglycan synthesis - Examples: β-lactams, bacitracin, vancomycin Mechanism of Action: Cell Membrane - Some antibiotics disrupt the integrity of cell Mechanism of action of the different class of membrane by binding to membrane antibiotics on protein synthesis are described phospholipids below: - Example: polymyxins Aminoglycosides - When aminoglycosides get their entry into the cytoplasm, inhibition of bacterial protein synthesis and mistranslation of protein occurs. They bind to the A-site of 16s rRNA with high affinity. Mistranslation occurs after the codon misreading on delivery of aminoacyl tRNA. This causes incorrect amino acids to change into polypeptides that result in cell damage. Some aminoglycosides inhibit protein synthesis by blocking in elongation stage of translation. Macrolides - For different modes were described in the mode of action shown by macrolides in Mechanism of Action: Protein Synthesis inhibition of bacterial protein synthesis. - Formation of 30S initiation complex - Assembly of 50S ribosome sub unit 1. During the early phase of translation - Formation of 70S ribosome from 30S and progression of peptide chain inhibition 50S occurs. - Elongation process 2. Peptidyl tRNA dissociation occurs from the ribosome. 3. Hindrance in peptide bond formation 4. Intervention with 50s subunit. In presence of some amino acid, macrolides were found by the ribosome which results in inhibition. And on some extent macrolides cause frameshifting causing rough bacterial protein synthesis. Chloramphenicol - Chloramphenicol affects the elongation steps in bacterial protein synthesis by inhibiting the peptidyl transferase activity. Chloramphenicol binds to A2451 and BSN 1A – BIOCHEM: ENZYMES AND VITAMINS  A2452 of ribosomal units that prevent Medical uses of enzyme peptide bond formation resulting in Enzyme also play an important role in inhibition of protein synthesis. treating disorders and balancing bodily conditions Tetracycline Particularly proteins such as enzyme, have - Tetracyclines are lipophilic molecules so the ability to accelerate chemical reactions they can cross the cell membrane. They are by serving as catalysts bacteriostatic. They bind to the 30s Blood serum enzyme activity tests may be ribosomal unit by preventing attachment of used to identify a wide range of illnesses transfer RNA which interferes with amino acids to form proteins and inhibit bacterial Blood enzyme assays used in the medical field protein synthesis. 1. Lactate dehydrogenase - is an enzyme that can be found in nearly all Oxazolidinone of the body’s cells and is involved in the - Oxazolidinones inhibit the formation of the production of energy initiation complex which is composed of mRNA, GTP, and other initiation factors. 2. Creatine phosphokinase They bind to the 50s ribosomal subunit. - muscular dystrophy may be detected with a Inhibition of formation of 70s subunit is by creatine phosphokinase, CPK, test, which is the after effect shown by binding to 50s the most accurate technique currently subunit complex. In the case of 70s available. formation translocation of peptide chain from P-site to A-site is inhibited which 3. Aspartate transaminase results in inhibition of protein synthesis. - is an enzyme that breaks down aspartate. Enzymes, which are proteins with specific Mechanism of Action: Transcription & DNA activities in the body, are required for nearly replication all of the body’s processes. - DNA synthesis: quinolones inhibit bacterial topoisomerase II Diseases that can be detected through the - RNA synthesis: rifampin inhibits bacterial help of enzymes RNA polymerase 1. Fatty liver - Antibiotic that affect mammalian cells: 2. Heart failure irinotecan, etoposide, doxorubicin 3. Osteoathritis - Actinomycin D: for cancer treatment Distribution and application of clinically important enzymes Mechanism of Action: Folic Acid synthesis - Bacteria synthesize their own folic acid - Example: sulfonamides and trimethoprim BSN 1A – BIOCHEM: ENZYMES AND VITAMINS  Vitamins - are an organic substances that are essential for carrying out the normal biochemical processes and physiological function of the body. - These are non-energy compounds and required in very small quantities - The vitamins generally served as a cofactor for the enzyme required in intermediary metabolism - The main source of vitamins is diet. A balanced diet supplies adequate amount of vitamins to fulfill the daily requirements The vitamins have been broadly divided into two groups 1. Fat soluble - vit. A,D,E, and K - These vitamins are stored by the body, mainly in the liver. Excessive intake of these can lead to toxicity due to accumulation, known as hypervitaminosis. Chemical name of vitamins Water-soluble Fat-soluble vitamins vitamins Vitamin B1 Vitamin A (Thiamine) (Retinoids) Vitamin B2 Vitamins D (Riboflavin) (Calciferol) Vitamin E Vitamin B3 (Niacin) (Tocopherol) Vitamin B5 Vitamin K (Pantothenic acid) Vitamin B6 (Pyridoxine) Vitamin B7 (Biotin) Vitamin B9 (Folic acid) Vitamin B12 (Cobalamin) Vitamin C (Ascorbic acid) 2. Water soluble - vit. B-complex group and vit. C - These vitamins are least stored and toxicity does not occur. The excess vitamins rapidly metabolized and readily excreted in the urine. BSN 1A – BIOCHEM: ENZYMES AND VITAMINS