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Lecture 10

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School
University of Toronto Scarborough
Department
Biological Sciences
Course
BIOC12H3
Professor
Shelley A.Brunt
Semester
Summer

Description
Lecture 10: Coenzymes and Vitamins Comparison of serine proteases  When we previously talked about: for determining enzyme composition/sequence we established that trypsin catalyzes the hydrolysis of peptide binds on the carbonyl side of arginine or lysine and chymotrypsin less specific on the carbonyl side of unchanged residues with aromatic or bulky hydrophobic side chains such as phenylalanine, tyrosine  Both trypsin and chymotrypsin contain a biding pocket that correctly positions the substrate for Nucleophilic attack by an active-site serine residue  Each enzyme has an extended region in which the substrate polypeptides fit  However the specificity pocket near the active-site serine is very different for each enzyme Binding sites of chymotrypsin, trypsin, and elastase (Slide 3)  The differing binding sites of these three serine proteases are primary determinants of their substrate specificities. (a) Chymotrypsin has a hydrophobic pocket that binds the side chains of aromatic or bulky hydrophobic amino acid residues. (b) A negatively charged aspartate residue at the bottom of the binding pocket of trypsin allows trypsin to bind the positively charged side chains of lysine and arginine residues. (c) In elastase, the side chains of a valine and a threonine residue at the binding site create a shallow binding pocket. Elastase binds only amino acid residues with small side chains, especially glycine and alanine residues. Serine Proteases Use Both the Chemical and the Binding Modes of Catalysis  Let’s examine the mechanism of chymotrypsin and the roles of three catalytic residues: His-57, Asp-102, and Ser-195. Many enzymes catalyze the cleavage of amide or ester bonds by the same process so study of the chymotrypsin mechanism can be applied to a large family of hydrolases. The catalytic site of chymotrypsin  The active site residues Asp-102, His-57, and Ser-195 are arrayed in a hydrogen-bonded network. The conformation of these three residues is stabilized by a hydrogen bond between the carbonyl oxygen of the carboxylate side chain of Asp-102 and the peptide- bond nitrogen of His-57. Oxygen atoms of the active-site residues are red, and nitrogen atoms are dark blue. Mechanism of chymotrypsin  SEE FIGURE 6.28 in Horton Review: Serine proteases use many catalytic modes  Steps 1-4: forward reaction used proximity effect, i.e. the gathering of reaction  Acid-base catalysis by histidine in steps 2 and 4 lower the energy barrier  Covalent catalysis using the –CH O2 of serine occurs in steps 2 through 5  The unstable tetrahedral intermediates at steps 2 and 4 are similar to the transition states for these steps  Hydrogen bonds in the oxyanion hole stabilize these intermediates  Serine proteases use both chemical modes (acid-base and covalent) and binding modes (proximity effect and transition state stabilization)  Site directed mutagenesis gas been used to show significance of the Ser-His-Asp catalytic triad:  In the bacterial serine protease subtilisin replacing the covalent catalyst serine or the acid-base catalyst histidine with alanine reduced enzymatic activity significantly  But even with mutation of all three amino acids to nonconserved amino acids the enzymatic rate was above the nonenzymatic rate due to the role of transition state stabilization  Cysteine, aspartyl and metalloproteases are other major peptide-cleaving enzymes  These are 3 alternate approaches to peptide bond hydrolysis (i.e. not based on activated serine residues)  Generate a nucleophile that attacks the peptide carbonyl group Cofactors and Co-enzymes  Many enzymes need cofactors for catalysis  Other chemical species, called cofactors, often participate in catalysis. Cofactors are required by inactive apoenzymes (proteins only) to convert them to active holoenzymes. There are two types of cofactors: essential ions (mostly metal ions) and organic compounds known as coenzymes. Essential ion cofactors  Over a quarter (25%) of all known enzymes require metallic cations to achieve full catalytic activity. These enzymes can be divided into two groups: metal-activated enzymes and metalloenzymes.  Metal-activated enzymes either have an absolute requirement for added metal ions or are stimulated by the addition of metal ions. Some of these enzymes require monovalent cations such as K+ and others require divalent cations such as Ca2+ or Mg2+. Kinases, for example, require magnesium ions for the magnesium-ATP complex they use as a phosphoryl group donating substrate. Magnesium shields the negatively charged phosphate groups of ATP making them more susceptible to nucleophilic attack.  Metalloenzymes contain firmly bound metal ions at their active sites. The ions most commonly found in metalloenzymes are the transition metals, iron and zinc, and less often, copper and cobalt. Metal ions that bind tightly to enzymes are usually required for catalysis. The cations of some metalloenzymes can act as electrophilic catalysts by polarizing bonds. For example, the cofactor for the enzyme carbonic anhydrase is an electrophilic zinc atom bound to the side chains of three histidine residues and to a molecule of water.  Example restriction endonucleases made by bacteria that cleaves foreign DNA are enzymes that require Mg2+ for catalytic activity  Many enzymes that act on phosphate-containing substrates require Mg2+  the ions of other metalloenzymes can undergo reversible oxidation: reduction by transferring electrons from a reduced substrate to an oxidized substrate  e.g. iron is part of the heme group of catalase which degrades hydrogen peroxide Coenzymes  Coenzymes can be classified into two types based on how they interact with the apoenzymes (the protein part minus the coenzyme).  Coenzymes of one type—often called cosubstrates—are actually substrates in enzyme- catalyzed reactions. A cosubstrate is altered in the course of the reaction and dissociates from the active site. The original structure of the cosubstrate is regenerated in a subsequent reaction catalyzed by another enzyme. The cosubstrate is recycled repeatedly within the cell, unlike an ordinary substrate whose product typically undergoes further transformation. Cosubstrates shuttle mobile metabolic groups among different enzyme- catalyzed reactions.  The second type of coenzyme is called a prosthetic group. A prosthetic group remains bound to the enzyme during the course of the reaction. In some cases the prosthetic group is covalently attached to its apoenzyme, while in other cases it is tightly bound to the active site by many weak interactions. Like the ionic amino acid residues of the active site, a prosthetic group must return to its original form during each full catalytic event or the holoenzyme will not remain catalytically active. Cosubstrates and prosthetic groups are part of the active site of enzymes. They supply reactive groups that are not available on the side chains of amino acid residues.  Every living species uses coenzymes in a diverse number of important enzyme catalyzed reactions. Most of these species are capable of synthesizing their coenzymes from simple precursors. This is especially true in four of the five kingdoms—prokaryotes, protists, fungi, and plants—but animals have lost the ability to synthesize some coenzymes.  Mammals (including humans) need a source of coenzymes in order to survive. The ones they can’t synthesize are supplied by nutrients, usually in small amounts (micrograms or milligrams per day). These essential compounds are called vitamins and animals rely on other organisms to supply these micronutrients. The ultimate sources of vitamins are usually plants and microorganisms. Most vitamins are coenzyme precursors—they must be enzymatically transformed to their corresponding coenzymes.  Two broad classes of vitamins have since been identified: water-soluble (such as B vitamins) and fat-soluble (also called lipid vitamins). Water-soluble vitamins are required daily in small amounts because they are readily excreted in the urine and the cellular stores of their coenzymes are not stable. Conversely, lipid vitamins such as vitamins A, D, E, and K, are stored by animals and excessive intakes can result in toxic conditions known as hypervitaminoses. ATP and Other Nucleotide Cosub
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