Introduction to Enzymes and Proteases

Overview of zymogens and the concept of protein digestion.
Transition into Chapter 7 covering kinetics and regulation.
Reminder regarding class schedule: No class on Friday due to fall break, but submission of discussion worksheet due.

Many types of proteases exist, each functioning effectively as enzymes.
Raises the question: Why don't proteases digest the proteins in cells?
- Proteins of interest:
- Enzymatic proteins
- Structural proteins (e.g., collagen, keratin)
Reason cells are not digested by proteases:
- Proteases are hydrolytic enzymes that catalyze hydrolysis reactions.
- These enzymes are synthesized as zymogens (inactive forms):
- Example includes pepsinogen, which is activated at low pH, preventing premature digestion of cellular proteins.

Definition: Inactive precursor forms of enzymes synthesized by cells.
Sequestration:
- Zymogens are stored in zymogen granules within the cell.
Activation Process:
- Zymogens undergo proteolytic processing to become active enzymes.
Example - Pepsinogen:
- Synthesized off the ribosome; contains a masking sequence that blocks the active site.
- Activation occurs at low pH leading to the removal of this masking sequence, thus exposing the active site.
Example - Chymotrypsinogen:
- Synthesized as a polypeptide of 245 amino acids, stabilized by five disulfide bonds.
- Requires cleavage by trypsin to form active chymotrypsin.
- Cleavage points: Between amino acids 15 and 16 (results in pi-chymotrypsin), further autocatalytic cleavage between amino acids 13-16 and 146-149 reveals the active site, which includes the substrate binding site and oxyanion hole.

Activation of chymotrypsinogen begins in the stomach:
- Low pH environment:
- Denatures proteins and activates pepsinogen to pepsin.
- Pepsin digests proteins into oligopeptides.
Hormonal Regulation:
- Oligopeptides trigger the release of two hormones:
- Secretin:
  - Promotes the release of sodium bicarbonate, neutralizing stomach acid in the intestine.
- Cholecystokinin (CCK):
  - Stimulates the release of pancreatic zymogens, stored in granules.
Trypsin Activation:
- Enteropeptidase catalyzes the conversion of trypsinogen to trypsin in the duodenum.
- Trypsin is the master activator, also activates proelastase, procarboxypeptidase, and pro lipase into their respective active forms.
- The action of trypsin is tightly regulated by pancreatic trypsin inhibitor which binds to trypsin with a very low dissociation constant of 0.1 picomolar, preventing unwanted activation of zymogens.

Proteolytic enzymes are synthesized as inactive zymogens and stored to prevent cellular deterioration.
A single trigger in the regulatory pathway (trypsin activation) implements full enzyme activation.
The pancreatic trypsin inhibitor limits enzyme activity until it is absolutely necessary for digestion.

Transitioning into enzyme kinetics and inhibition.
Importance of studying kinetics to understand reaction mechanisms, mutations in metabolic pathways, and biochemical regulation.

Heat: Increases the rate of enzymatic reactions.
pH: Affects enzyme activity due to changes in ionization of amino acids.
Concentration:
- The concentration of both enzyme and substrate highly influences the reaction rate.
Rate Law Equation for a 2nd Order Reaction:
- $v = k_{1} imes [E] imes [S]$
This equation encompasses the significant influences of enzyme concentration and substrate concentration on reaction velocity, alongside pH and buffer conditions.

The rate of reaction can be monitored:
- By observing the disappearance of substrate: $\Delta [S]/\Delta t$
- By measuring the formation of product: $\Delta [P]/\Delta t$
Reaction velocity versus time plots showcase typical enzyme catalyzed reactions, initially high in substrate concentration, transitioning over time.
Initial Velocity (v0): Defined as the formation of product per unit time at the beginning of the reaction before significant substrate depletion occurs.

Initial rates lead to simplifications in kinetic schemes.
Key steps include:
- Formation of the enzyme-substrate complex (ES).
- Conversion from ES to product (dependent on rate constant k2).
- Ignoring back-association of the enzyme-substrate complex to simplify calculations.

As substrate concentrations increase, initial reaction rates rise until reaching saturation (Vmax).
Vmax is defined as the maximum rate of reaction uninfluenced by substrate concentration, reliant on the enzyme concentration and the rate constant kcat.
Kilometers (Km):
- Defined as the substrate concentration that corresponds to half of Vmax, an important characteristic of enzyme kinetics.

Given Vmax is observed or estimated from a plot:
- Example Vmax = 35 micromoles per second as a derived figure.
To estimate Km:
- Applying the Km definition: Km = [S] at 1/2 Vmax.
- For Vmax = 35, Km would be where reaction rate is measured as 17.5 micromoles, yielding approximate concentration at that point from a graph or data set.

To determine the turnover number (kcat), the enzyme concentration must be known.
Calculating the fraction of enzyme active sites bound to substrate can be assessed at varying substrate concentrations, revealing insights into enzyme kinetics.

Introduction of scientific pioneers Michaelis and Menten and their contributions to understanding enzyme-substrate kinetics and the formation of intermediate enzyme-substrate complexes.
The Michaelis-Menten equation depicting the relationship between reaction rate and substrate concentration takes the form of a rectangular hyperbola: $\frac{V<em>{max} \cdot [S]}{K</em>m + [S]}$
Variable definitions:
- Vmax: Maximal asymptote (maximum reaction rate)
- Km: Reflects the substrate concentration at which velocity is half-maximal

The derivation will break down how to connect kinetic variables through strategic assumptions (steady-state) and mathematical relationships to yield the Michaelis-Menten expression.
Importance of practice: Familiarity with the derivation process is crucial.

Students are encouraged to clarify any uncertainties pertaining to course materials before the next class session after the fall break.
Review all discussed topics to solidify understanding of enzyme kinetics and proteolytic regulation ahead of examinations.