Enzymes 4 - Cofactors and Proteases PDF

Chemical Biology and Enzymes CHEM3012 2024/25 Enzymology 4 – Cofactors and Proteases Ellis O’Neill Office BDI A13 Ellis.O’[email protected]. uk  Learning outcomes  Further reactions with coenzymes  Know the molecular mechanism of action of...

Chemical Biology and Enzymes CHEM3012 2024/25 Enzymology 4 – Cofactors and Proteases Ellis O’Neill Office BDI A13 Ellis.O’[email protected]. uk  Learning outcomes  Further reactions with coenzymes  Know the molecular mechanism of action of serine proteases  Know the molecular mechanism of peptide bond cleavage by cysteine proteases  Know molecular mechanism of peptide bond cleavage by acid proteases  The use of proteases CHEM3012 – Enzymes 4 Ellis O’Neill 2  Coenzyme – Cobalamin  Cobalamin  Vitamin B12 O O H2N H 2N N N O OH H 2N N N HO O Co O P O R O- N O N N H H 2N NH2 H O NH2 O CHEM3012 – Enzymes 4 Ellis O’Neill 3 O  Coenzyme – Cobalamin  Cobalamin  Vitamin B12  Can shuttle between CoI, CoII and CoIII O O H2N H 2N N N O OH H 2N N N HO O Co O P O R O- N O N N H H 2N NH2 H O NH2 O CHEM3012 – Enzymes 4 Ellis O’Neill 4 O  Coenzyme – Cobalamin  Cobalamin  Vitamin B12  R can be a range of groups including CN, HO and Me O O H2N H 2N N N O OH H 2N N N HO O Co O P O RN N N O O- H H 2N NH2 H O NH2 O CHEM3012 – Enzymes 4 Ellis O’Neill 5 O  Coenzyme – Cobalamin  Cobalamin  Vitamin B12  R can be a range of groups including CN, HO and Me  E.g. methionine synthase SH Me S MeB12 B12 OH OH H2N H2N O O CHEM3012 – Enzymes 4 Ellis O’Neill 6  Coenzyme – Biotin  Biotin  Vitamin B7 O O NH H HN OH H S CHEM3012 – Enzymes 4 Ellis O’Neill 7  Coenzyme – Biotin  Biotin  Vitamin B7  Transfer of carboxylate  E.g. pyruvate carboxylase O O O N NH H H S O OH CHEM3012 – Enzymes 4 Ellis O’Neill 8  Coenzyme – PLP  Pyridoxal phosphate (PLP)  Vitamin B6 O O HO P O OH HO N CHEM3012 – Enzymes 4 Ellis O’Neill 9  Coenzyme – PLP  Pyridoxal phosphate (PLP)  Vitamin B6  Forms Schiff base with amines, including enzyme active site Enzyme Lys N O HO P O OH HO N CHEM3012 – Enzymes 4 Ellis O’Neill 10  Coenzyme – PLP  Pyridoxal phosphate (PLP)  Vitamin B6  Forms Schiff base with amines, including enzyme active site  Carries out amino transferases, eliminations and isomerisations R HO NH2 O O O HO P O OH HO N CHEM3012 – Enzymes 4 Ellis O’Neill 11  Proteases  Proteases (peptidases) cleave the peptide bond between amino acids  Exopeptidases: cleave the terminal peptide bonds releasing an amino acid and reducing the chain length by one  Endopeptidases: cleave internal peptide bonds releasing two peptides  The overall reaction is a hydrolytic reaction CHEM3012 – Enzymes 4 Ellis O’Neill 12  Proteases  Proteases (peptidases) cleave the peptide bond between amino acids  The overall reaction is a hydrolytic reaction  Protease families based upon their mechanism of action:  Serine proteases  Cysteine proteases  Acid proteases  Metalloproteases CHEM3012 – Enzymes 4 Ellis O’Neill 13  Serine proteases  The family name is derived from a conserved serine residue in the active site which participates in the reaction mechanism  The active site contains a catalytic triad:  Aspartate to align the histidine and act as general acid  Histidine to activate the serine and act as a base  Serine to act as nucleophile OH O O OH N NH HN Asp His Ser Substrate CHEM3012 – Enzymes 4 Ellis O’Neill peptide 14  Serine proteases Asp His Ser CHEM3012 – Enzymes 4 Ellis O’Neill 15  Serine proteases  The family name is derived from a conserved serine residue in the active site which participates in the reaction mechanism  The active site contains a catalytic triad  The cleaving reaction proceeds in two steps:  Formation of an acyl-enzyme intermediate involving the reactive Ser residue in the active site  Hydrolysis of the acyl-enzyme intermediate CHEM3012 – Enzymes 4 Ellis O’Neill 16  Mechanism of serine proteases O N R H OH N NH O O Enzyme Ser Enzyme Enzyme His Asp CHEM3012 – Enzymes 4 Ellis O’Neill 17  Mechanism of serine proteases O R O N NH HO O CHEM3012 – Enzymes 4 Ellis O’Neill 18  Protease selectivity  Most serine proteases exhibit no absolute substrate specificity.  Polypeptides bind to the enzyme through their main chain atoms which form H-bonds in a short antiparallel b-sheet with the main chain atoms of a loop region in the enzyme CHEM3012 – Enzymes 4 Ellis O’Neill 19  Protease selectivity  Most serine proteases exhibit no absolute substrate specificity.  Polypeptides bind to the enzyme through their main chain atoms which form H-bonds in a short antiparallel b-sheet with the main chain atoms of a loop region in the enzyme  Many serine proteases show a preference for a particular side chain before the cleaved peptide bond Scissile bond Substrate N R1 R2 R3 R4 R5 R6 R7 C Protease Preferred side chain CHEM3012 – Enzymes 4 Ellis O’Neill 20  Protease selectivity  Most serine proteases exhibit no absolute substrate specificity.  Polypeptides bind to the enzyme through their main chain atoms which form H-bonds in a short antiparallel b-sheet with the main chain atoms of a loop region in the enzyme  Many serine proteases show a preference for a particular side chain before the cleaved peptide bond  Controlled by the selectivity pocket CHEM3012 – Enzymes 4 Ellis O’Neill 21  Selectivity pocket H O Preference for positively N N H charged amino acids O O NH3 HN NH O O N H O Trypsin Aspartate Negative charge CHEM3012 – Enzymes 4 Ellis O’Neill 22  Selectivity pocket H O H O Preference for positively N N N N bulky amino acids H H O O O O NH3 HN NH HN NH O HO OH O N N H H O O Trypsin Chemotrypsin Aspartate Serine (Negative charge) (Small) CHEM3012 – Enzymes 4 Ellis O’Neill 23  Selectivity pocket Preference for small O O non-polar amino acids H H N N O N N H H H N N H O O O O O O NH3 HN NH HN NH O HN HO NH HO OH O N H N N O H H O O Trypsin Chemotrypsin Elastase Aspartate Serine Valine/Threonine (Negative charge) (Small) (Bulky) CHEM3012 – Enzymes 4 Ellis O’Neill 24  Cysteine proteases  They utilise an active cysteine residue in the active site  eg papain, ficin, bromelain  Because the pKa of the Cys side chain (1.92)​it is a much stronger acid than the side chain of Ser (pKa = 13), there is no need for an Asp acid  instead of a catalytic triad they use a His, Cys catalytic dyad CHEM3012 – Enzymes 4 Ellis O’Neill 25  Cysteine proteases CHEM3012 – Enzymes 4 Ellis O’Neill 26  Cysteine proteases  Mechanism of action of papain:  Catalytic dyad:  Cys25 (nucleophilic attack)  His159 (general acid/base)  Oxyanion hole  Cys25, Gln19 stabilize the oxyanion via H-bonding interactions  Substrate binding:  Binding site accommodates 7 amino acids. CHEM3012 – Enzymes 4 Ellis O’Neill 27  Mechanism of Papain Enzyme Gln H 2N O O N R H S HN NH Enzyme Cys Enzyme His CHEM3012 – Enzymes 4 Ellis O’Neill 28  Aspartyl proteases  As the name implies they employ reactive Asp residues in the active site  eg pepsin, chymosin, HIV-protease etc  The cleaving reaction proceeds through general acid-base catalysis CHEM3012 – Enzymes 4 Ellis O’Neill 29  Aspartyl proteases Enzyme Asp HO O O N R H HO H O O Enzyme Asp CHEM3012 – Enzymes 4 Ellis O’Neill 30  Biotechnology  Cleaning agents eg biological detergents  Meat tenderiser eg pineapple steak  Cheese making eg rennet and vegetarian rennet CHEM3012 – Enzymes 4 Ellis O’Neill 31  Biotechnology  Cleaning agents eg biological detergents  Meat tenderiser eg pineapple steak  Cheese making eg rennet and vegetarian rennet  Protein engineering eg TEV protease  Cleaves at a specific sequence, removing tags TEV Tag Tag Enzyme Enzyme CHEM3012 – Enzymes 4 Ellis O’Neill 32  Biotechnology  Cleaning agents eg biological detergents  Meat tenderiser eg pineapple steak  Cheese making eg rennet and vegetarian rennet  Protein engineering eg TEV protease  Protein fingerprinting – proteomics  Protease cleaves at specific positions, based on specificity pocket CHEM3012 – Enzymes 4 Ellis O’Neill 33  Biotechnology  Protein fingerprinting – proteomics  Protease cleaves at specific positions, based on specificity pocket IVGGYTCEENSLPYQVSLNSGSHFCGGSLISEQWVVSAAHCYKTRIQVRLGEHNIKVLEGNEQFINAVKIIRHPKYNRDT LDNDIMLIKLSSPAVINARVSTISLPTAPPAAGTECLISGWGNTLSFGADYPDELKCLDAPVLTQAECKASYPGKITNSMFC VGFLEGGKDSCQRDSGGPVVCNGQLQGVVSWGHGCAWKNRPGVYTKVYNYVDWIKDTIAANS Trypsin IVGGYTCEENSLPYQVSLNSGSHFCGGSLISEQWVVSAAHCYK CLDAPVLTQAECK TRIQVRLGEHNIK ASYPGKITNSMFCVGFLEGGK VLEGNEQFINAVK DSCQRDSGGPVVCNGQLQGVVSWGHGCAWK IIRHPK NRPGVYTK YNRDTLDNDIMLIK VYNYVDWIK LSSPAVINARVSTISLPTAPPAAGTECLISGWGNTLSFGADYPDELK DTIAANS CHEM3012 – Enzymes 4 Ellis O’Neill 34  Learning outcomes  Further reactions with coenzymes  Know the molecular mechanism of action of serine proteases  Know the molecular mechanism of peptide bond cleavage by cysteine proteases  Know molecular mechanism of peptide bond cleavage by acid proteases  The use of proteases CHEM3012 – Enzymes 4 Ellis O’Neill 35 Chemical Biology and Enzymes CHEM3012 2024/25 Enzymology 4 – Cofactors and Proteases Ellis O’Neill Office BDI A13 Ellis.O’[email protected]. uk