Catalytic Mechanisms I Flashcards
Name the 6 catalytic mechanisms
Acid-base
Covalent
Metal ion
Electrostatic
Proximity & orientation
Preferential transition state binding
What does catalysis result in?
Lowers the free energy of activation (ΔG#)
What is an example of an acid-base catalyst?
RNase A = cleaves RNA molecule at the phosphodiester bond
What reaction does RNase A catalyse and how?
Cleaves RNA molecule at the phosphodiester bond
Hydrolysis reaction
What is the intermediate and second substrate of the RNase reaction?
2,3-cyclic nucleotide = intermediate
H2O = substrate
Hydrolysis reaction = cleaves intermediate, leading to final product
What special group does RNase A have at its active site?
Two histidine residues, which have imidazole groups
Imidazole groups can function as both base and acid
What does the Hendeerson-Hasselbalch equation say?
pH = pKa + log [A-]/[HA]
What happens when acid and base conc are equal?
pH = pKa
What does pKa tell us?
pKa value is one method used to indicate the strength of an acid.
pKa is the negative log of the acid dissociation constant or Ka value.
A lower pKa value indicates a stronger acid
What does covalent catalysis involve?
TRANSIENT formation of catalyst-substrate covalent bond
Give an example of covalent catalysis
Nucleophilic attack
Nucleophile (enzyme) attacks electrophile (substrate) and transient covalent bond formation results
Define nucleophiles and electrophiles
Nucleophile = electron rich
Electrophiles = electron poor
Give examples of nucleophiles
electron RICH
S
O
N
AND imidazole groups
What reaction does pyruvate decarboxylase catalyse and what is needed?
Pyruvate to acetaldehyde (alcohol fermentation)
Requires COFACTOR = thiamine pyrophosphate (TPP)
What is the role of thiamine pyrophosphate in decarboxylation?
TPP has thiazolium ring (nucleophile) attacks carbonyl C of pyruvate
CO2 leaves = generates carbanion
Protonation of carbanion
Elimination of TPP to form acetyldehyde and regenerate active enzyme
What is the difference between metal-activated enzymes and metalloenzymes?
Metal-activated enzymes = loosely bind metal ions
Metalloenzymes = contain tightly bound transition metal ions
What is the role of Mg2+ in relation to ATP?***
Shield negative charge
Can form 6 coordinate bonds = so can be involved in interaction with enzymes
What is the role of Zn2+ in liver alcohol dehydrogenase?
Role in orientation/polarization of ETHANOL (substrate)
OH- is attracted to Zn2+ so it sits in the active site in the correct orientation
What is the role of Zn2+ in carbonic anhydrase?
CO2 + H2O > H2CO3
Zinc is tetrahedrally coordinated to 3 His side chains and then water molecule
Zinc helps to polarize the water
What is the His residue not bonded to Zn2+ and its ROLE?***
His 64 part of active site
How does electrostatic catalysis work?
Increases reaction rate by arrangement of charge distribution around active site
Stabilizes the transition state of the catalyzed reaction
May so guide polar substrates to their binding sites = increase rate of reaction
What effect do enzymes have on proximity and orientation?
Bring substrates into contact w catalytic group/other substrates
Bind substrate in right orientation
Freeze out translational and rotational motions of substrates/catalytic groups
Give an example of enzyme that effects proximity and orientation
Adenylate kinase = part of the nucleoside monophosphates (NMP)
What form do Nucleoside Monophosphate exist in within the cell?
Mg2+ - NMP
Without metal ion = NO enzyme activity
What is the structure of adenylate kinase?
P-loop and lid domain
What conformational change occurs when adenylate kinase binds ATP?
P-loop closes down and interacts with beta P group
Gamma P group is thus positioned next to binding site of second substrate
What is preferential transition state binding?
Enzymes bind the transition state of the reaction with greater affinity than its substrate
Give an example of an enzyme with preferential transition state binding
Proline racemase
What are the 5 approaches to determine enzyme catalytic mechanisms?
Kinetic studies
Intermediates
X-ray crystallography
Chemical modifications of side chains
Site-directed mutagenesis
What is chymotrypsin?
Digestive enzyme classified as a serine protease, primarily found in the small intestine.
It catalyzes the hydrolysis of peptide bonds in proteins
How was chymotrypsin’s intermediate used to determine catalytic mechanism?
Acyl-enzyme intermediate exists for very short time
Can manipulate conditions to isolate intermediate and do X_ray crystallography study
Found out that side chain of Ser195 = acylated
What is chymotrypsin catalytic triad?
His 57
Asp 102
Ser 195
What are the 3 serine proteases?
Chymotrypsin
Trypsin
Elastase
What residues are the serine proteases specific for?
Chymotrypsin = bulk hydrophobic
Trypsin = positively charged residues
Elastase = small neutral residues
Describe the substrate binding pockets of serine proteases
Chymotrypsin = small side chains to fit the bulky residues
Trypsin = negative residues (Asp) to attract positive residues
Elastase =
What is used in affinity labelling?
Substrate analog
Bearing reactive group that specifically binds at the enzyme active site
AND forms stable covalent bond with enzyme
What is affinity labelling used for?
What is TPCK?
Affinity label for chymotrypsin
How does TPCK affect chymotrypsin?
TPCK will inactivate chymotrypsin because it will make covalent bonds at active site
How do you inhibit the labelling of an enzyme?
Adding substrate or competitive inhibitor will inhibit the labelling of TPCK
What evidence proves that a modified side chain is at the active site?
Linear relationship between extent of modification and inactivation
Addition of substrate must protect against inactivation
What are the pre-requisites for site-directed mutagenesis?
Gene/ORF coding for the protein is available
Suitable system for expression of the gene/ORF
What are the steps of site-directed mutagenesis
3D structure is known or can be predicted
Using structure = select residues that may be important in catalysis
Mutate these residues and determine the effect on activity of the enzyme
What are the two types of site-directed mutagenesis?
Conserved mutation = replace with amino acid with similar properties
Non-conserved mutation = replace with amino acid with different properties
What bonds does chymotrypsin cleave?
Both ester and amide bonds
What is the reaction of p-Nitrophenylacetate and chymotrypsin?
p-nitrophenylacetate + chymotrypsin
Products = pNP and acyl-enzyme intermediate
Split the acyl-enzyme intermediate = acetate and enzyme
Describe the two kinetic phases
Burst phase = rapid formation of p-NP and correlates to amount of active enzyme
Steady-state = pNP is generated at a reduced but constant rate
