9 - Lecture Guide

Review of Lecture 9 Questions to think about…. Know that the enzymatic cleavages of amide or ester bonds by nucleophilic attack are important in biological systems. Which enzyme was the first protease catalytic site to be investigated? ALPHA CHYMOTRYPSIN, RECALL THAT CATALYTIC TRIADS ARE IMPORTANT ENZYMATIC REACTIONS THAT CLEAVE AMIDE AND ESTER LINKAGES BY NUCLEOPHILIC ATTACKS. What is a salt bridge? A BRIDGE BETWEEN ASP AND HISTIDINE VIA HYDROGEN BONDING? What is a catalytic triad? COMPOSED OF THREE AMINO ACIDS MAKING UP THE ACTIVE SITE, ASPARTATE, HISTIDINE, AND SERINE What do ?-chymotrypsin, trypsin and elastase have in common? THE ARE ALL ZYMOGENS THAT ARE CONVERTED INTO ACTIVE PANCREATIC ENZYMES VIA THE ENZYME ENTEROPEPTIDASE. THE DIFFERENCE BETWEEN THE ZYMOGEN (PROENZYME) AND THE ACTICE FORM IS THAT THE ZYMOGEN HAS A HYDROPHOBIC BINDING POCKET WHICH IS LOST UPON CONVERSION. Know that the synergistic nature of the side chains in the catalytic site enhance reaction rate. WHEN SERINE IS ACCOMPANIED BY HISTIDINE AND ASPARTATE, SERINE ACTING AS AN ACID CAN BE VERY EFFECTIVE. What method is used to investigate the structure of the catalytic site? Nucleophilic attacks are directed at which atom? Lipases may have covergently evolved a catalytic triad. The attacking and leaving atoms are reversed. What does this suggest about the catalytic mechanism of lipases? What types of residues are proposed to be necessary in the active site? How would penicillin acylase and ?-lactamase given that the substrates are structurally similar yet the enzymes cleave at a different site? THIS IS BECAUSE THEY BOTH HAVE A DIFFERENCE IN THEIR ACTIVE SITE DUE TO A DIFFERENT CONFIGURATION OF AMINO ACIDS THAT CAN BREAK CERTAIN REGIONS. Know that Ntn hydrolases have an N-terminal nitrogen that acts like a base. AND A NUCLEOPHILE. Know that the ?-lactamase breaks open the ?-lactam ring in penicillin while penicillin acylase removes the side group Consider the variations in the serine catalytic triad. Know that the catalytic nucleophile can also be a cysteine residue instead of a serine. What did Radisky and Koshland set out to investigate in their paper? How did they come up with the clogged gutter mechanism? What does ‘clogged gutter’ mean in the context of the active site? THE SET OUT TO INVESTIGATE HOW THE CI2 INHIBITS THE SUBSTILISIN ENZYME, I.E. IS IT DUE TO THE STRONG HOLD OR FIT OF THE ENZYME THAT CAUSES EXTREME RIGIDITY AND IN TURN PREVENT NUCLEOPHILIC ATTACKS FROM TAKING PLACE, … POOR ORIENTATION … POSITIONING OF THE LEAVING GROUP FAVOURS THE BACK REACTION? THE ARREST OF ENZYMATIC REACTION AT THE INTERMEDIATE STAGE ALLOWED THEM TO DETERMINE THAT THE CONSENSUS NUCLEOPHILIC ATTACK ANGLE IS CLOSE TO 90° IN THE REACTIVE MICHAELIS COMPLEXES. THE CLOGGED GUTTER MEANS THAT A COMBINATION OF A NETWOEK OF HYDROGEN BONDS, AN ACYL ENZYME, AND THE ORIENTATION OF A RELIGATING ENZYME ALL PLAY A ROLE IN ARRESTING A REACTION, EACH FACTOR ON THEIR OWN IS INSUFFIENCT. If the serine proteases (trypsin, chymotrypsin, and elastase) contain the same catalytic triad, why do they catalyze cleavage of only those peptide bonds in which the carbonyl group is donated by specific amino acid residues? What is the function of enteropeptidase? IT CONVERTS TRYPSINOGEN INTO TRYPSIN. Pancreatic trypsin inhibitor binds to and inactivates trypsin formed in the pancreas. Since trypsin inhibitor is itself a protein, why is it not digested by trypsin? Understand how serine proteases cleave peptide bonds. Understand how lysozyme hydrolyzes polysaccharide chains. How might lysozyme be useful for a biochemist studying proteins? THIS OCCURS WHEN THE SUGAR ENTERS THE CLEAVAGE SITE. THE GLUTAMINE ACTS AS A GENERAL ACID WHERE IT DONATES A HYDROGEN TO THE CLEAVAGE PORTION AND THE ASPARTATE RESIDUE IS IN ITS IONIZED STATE AND STABALIZES THE SUGAR THAT IS BEING CLEAVED. Who was Daniel Koshland, Jr? HE IS THE GUY WHO DISCOVERED THAT WHEN GLUCOSE ENTERS THE HEXOKINASE ENZYME, IT PROHIBITED ATP FROM BECOMING HYDROLYZED. For extra review do the following questions from your text: Page 226: #50, #51, #54