CHEM 271: Enzyme Kinetics and Inhibition

• CHEM 271 Biochemistry I, Lecture 6 covers enzyme kinetics, enzyme mechanisms, and enzyme regulation.

Enzyme Activity

• An enzyme's compliance with Michaelis-Menten kinetics, Michaelis constant (K_M_), and maximal velocity (V_max_) can all be determined from provided data.
• Type I inhibitor can also be determined.

Enzyme Inhibition

• Eadie-Hofstee plots can be used to look at enzyme inhibition.
• Different types of enzyme inhibition include competitive, uncompetitive, and noncompetitive inhibition.

Chymotrypsin

• Chymotrypsin has a catalytic triad consisting of His57, Asp102, and Ser195
• Substrate binding is when the side chain of the residue nestles adjacent to the cleaving peptide bond in a hydrophobic pocket.
• Ser and His's interaction generates a strong nucleophilic alkoxide ion on Ser, which attacks the peptide carbonyl group, forming an acyl-enzyme.
• Instability of the charge on the substrate carbonyl oxygen leads to collapse of the tetrahedral intermediate

Proteases

• Proteases have specificity
• Proteases are activated and inactivated.
• Proteases are important in blood clotting.

Other Types of Proteases

• Cysteine proteases rely on a Cys residue, activated by a His residue, to act as a nucleophile, attacking the peptide bond.
• Aspartyl proteases use a pair of Asp residues, acting together, to enable a water molecule to attack the peptide bond.
• Metalloproteases use a bound metal ion, typically zinc, to activate a water molecule to act as a nucleophile, attacking the peptide carbonyl group.

Protease Inhibitors

• Protease inhibitors block protease activity.
• Captopril inhibits metalloprotease ACE and regulates blood pressure.
• Indinavir (Crixivan) is an inhibitor of HIV protease (an aspartyl protease) that cleaves vital proteins into their active forms.
• HIV protease functions as a dimer with identical subunits and has an active site aspartate residue from each chain.
• After the substrate binds, the flaps close down on the binding pocket.

Regulatory Strategies

• Allosteric control works by regulating molecules
• Isozymes are enzymes that catalyze same reaction but show different catalytic and regulatory properties.
• Reversible covalent modification is a type of temporary modification.
• Proteolytic activation involves irreversible proteolytic cleavage to activate an enzyme.
• The control amount of enzyme that is present is also an important regulatory step.

Allosteric Control

• Committed steps, that are irreversible, are catalyzed by allosteric enzymes.
• Products of the reaction are committed to the end synthesis products.
• Aspartate transcarbamoylase (ATCase) catalyzes the first step in pyrimidine biosynthesis.
• ATCase is controlled by cytidine triphosphate (CTP)
• Most allosteric enzymes show cooperativity in steady-state kinetics.
• ATCase contains 6 Catalytic subunits and 6 regulatory subunits.
• Two catalytic dimers are stacked upon each other, linked by 3 regulatory dimers.

PALA

• PALA is a potent competitive inhibitor
• PALA is bisubstrate analog, representing an intermediate during the ATCase catalytic pathway.
• ATCase's active site is compromised of multiple subunits and binds to to allosteric sites.
• PALA alters ATCase by inducing a T state to R state conversion.
• CTP (allosteric inhibitor) binds to create a more stabilized state, which limits reaction speed.
• ATP can be an allosteric activator, to increase product formation.

Isozymes

• Isozymes are tissue-specific and vary in regulatory properties.
• Lactate dehydrogenase (LDH) is a tetrameric protein that catalyzes a step in anaerobic glucose metabolism and glucose synthesis.
• Muscle (M) and Heart subunits are 75% identical.

Covalent Modification

• Covalent modifications include phosphorylation, acetylation, myristoylation, ADP ribosylation, farnesylation, γ-Carboxylation, sulfation, and ubiquitination, and regulate protein activity.

Protein Kinases & Protein Phosphatases

• Protein kinases catalyze phosphorylation of proteins
• Protein phosphatases catalyze de-phosphorylation
• Kinases adds a phosphate group, while phosphatases remove a phosphate group.
• ATP hydrolysis releases 12 kcal mol^-1 or 50 KJ mol^-1
• Phosphorylation and dephosphorization do not occur independently, and are catalyzed by individual enzymes.
• Many different enzymes are activated by serine and threonine.
• Protein Kinase A is also activated by cAMP.
• Protein Kinase activates intracellular protein that stimulates ATP.

Proteolysis

• Digestive Proteases and Zymogens can act as precursers.
• A Zymogen are inactive precursers
• Digestion requires several different types of proteases.
• Trypsinogen undergoes enterpepsidase-> Trypsen
• Beta chain N terminus interacts with C-chain side chain through elecrostatic interactions.