Which amino acids are good for acid and base catalysis?
Acids: Glu, Asp
Weak Acids: Tyr, Cys
Bases: His, Lys
Where does RnaseA cut in the strand?
in the middle
hydrolyze a phosphodiester bond found in RNA
What does the Lys and Arg residues accomplish in RnaseA?
stabilizes negative charge (RNA backbone, transition state)
What happens in the first step of the RnaseA mechanism?
His 12 acts as a base and His 119 is a general acid to promote nucleophilic attack and bond cleavage
What is the transition state of this first part of the RnaseA mechanism?
pentavalent TS, phosphate is bonded to five things
How does Lysine structures stabilize the RnaseA mechanism?
the transition state is very negative because of phosphate binding to five things
What is the intermediate of the RnaseA reaction?
2’,3’ cyclic nucleotide
What are good nucleophiles for covalent catalysis?
RO-
RS-
Histidine imidazole group
What are structural and catalytic ions for metal ion catalysis?
Structural ions: Na+, K+, Ca2+
Catalytic ions: transition metals
What does the metal ions do in the metal ion catalysis?
Bind substrates to orient themselves, mediate redox reactions, stabilize/shield charge, interact with water to produce nucleophiles
What can enzymes do in terms of proximity?
Can orient reaction and catalytic groups to maximize interaction, may be charged to stabilize transition states
What does enzymes bind to the most (reactants, products, transition state)?
transition state to stabilize it and reduce free energy
What type of catalysis is carbonic anhydrase?
metal ion catalysis
What is the metal ion in carbonic anhydrase and how does it act?
Zn2+, ionizes H2O to create OH- that nucleophilic attacks the CO2 to create HCO3-
What does carboxypeptidase cleave?
Hydrolyzes an amide bond in proteins, removes the C terminus with bulky hydrophobic side chain
What does the C terminus of the substrates interact with in carboxypeptidase?
Arg- electrostatic interaction
two hydrogen bonds with other molecules
How to count the carbonyl groups in the substrate and what do they do in carboxypeptidase?
-1, -2 (count backwards from the C-terminus carbonyl)
forming hydrogen bonds with arginine
What does the hydrophobic pocket do in carboxypeptidase?
Demonstrates that induced fit in a enzyme (Tyr248) and orients the substrate. Moves 12 A.
What is the purpose of zinc in carboxypeptidase?
Acidify the water, stabilizes Oh- nucleophile, and stabilizes oxyanion TS
What does Glu270 do in carboxypeptidase?
acts as a general base, deprotonates water and help create the nucleophilic OH- molecule
What is the transition state of carboxypeptidase
negative, tetrahedral, oxyanion
stabilized by arginine residues
Where does chymotrypsin hydrolyze at?
hydrolyze peptide bonds on their c-terminal side
What are the three serine proteases and where do they cut at (specificity pocket)?
Chymotrypsin: bulky hydrophobic
Trypsin: Lys/Arg
Elastase: Ala/Gly
How many domains are in chymotrypsin?
2 domains, highly conserved
