L4: Enzymology

Question 1

What are enzymes?

Answer 1

Proteins with catalytic properties in which they can accelerate the rates of chemical reactions
•Most enzymes exhibit absolute reaction specificity

Catalytic activity is dependent on multiple factors
•Proper protein structure and folding
•Available and appropriate substrate
•Sufficient enzyme (concentration and active sites)
•Optimal environmental conditions (temperature, pH)

Question 2

Describe Protein Structure.

Answer 2

Primary structure
•Simplest level of structure; polypeptide sequence

Secondary structure
•Intra-polypeptide structures
•α helix, β pleated sheet

Tertiary structure
•Inter-sidechain structures due to –R group interactions
•Still within same overall polypeptide chain

Quarternary structure
•Inter-polypeptide structure from non-covalent interactions

Question 3

Describe biological catalysis and models to characterize substrate binding.

Answer 3

Protein structure determines (in part) substrate selectivity:

1. Active site(s) are relatively small compared to enzyme molecule
   •Typically occur in clefts and crevices within protein
   •Spatial arrangement in active site designed to complement intended substrate(s)
2. Models have been developed to characterize substrate binding
   •“Lock and key” model describes spatial complementarity to promote enzyme specificity but does not explain stabilization of transition state
   •Induced fit model enhances previous model by describing unique conformation of enzyme-substrate complex achieved only upon substrate binding

Question 4

Describe energetics of catalysis in terms of free energy of activation (deltaG).

Answer 4

Enzymes accelerate reactions via decreasing free energy of activation (ΔG‡)

•ΔG‡ (also known as Gibbs free energy) refers to energy absorbed by substrate(s) required for conversion to product(s)

(see slide 8 figures)

Question 5

List factors affecting enzymatic activity:

Answer 5

• pH
• Temperature
• Activators
• Inhibitors
• Salt and protein concentrations

Question 6

Describe the effect of pH of enzyme catalysis.

Answer 6

Many enzymes display maximal catalytic activity between pH 7-8
•Reflects overall physiological pH when there are no underlying acid-base disorders
•pH extremes at either end can denature enzymes

Some enzymes have evolved to function at more extreme pH levels
•Pepsin is maximally active in acidic conditions (pH 1.5-2)
•Alkaline phosphatase is maximally active in alkaline conditions (pH 9-10)

pH conditions can be manipulated to achieve enzyme selectivity
•Enzyme isoform differentiation (LDH reaction influence by pH, see slide 10)
•Forcing reaction directions

Question 7

Describe the effect of temperature on enzyme catalysis.

Answer 7

Enzymes generally have an optimal temperature of 37ºC
•Activity increases with temperature but denaturing will inevitably occur

Most analytical systems operate at 37ºC to reflect physiological conditions
•Reference methods for measured enzymes are developed at 37ºC
•Accurate temperature control on analytical systems is critical (+ 0.1ºC)

Temperature can be used to modulate enzymatic activity
•Cold storage for preservation
•Heat stability for isoform differentiation

Question 8

Describe the effects of activators/cofactors.

Answer 8

Activators increase rate of enzyme reactions through promoting active state of enzyme and/or substrate
•Can be inorganic ions (cofactors) or organic molecules (coenzymes)
•Activators bind to apoenzyme to create holoenzyme

Many enzymes contain cofactors within their structure
•Mg2+ is essential for creatine kinase activity

Question 9

Describe the effect of inhibitors on enzyme catalysis.

Answer 9

Enzyme inhibition can be reversible or irreversible
•Irreversible inhibition usually involves covalent bond between inhibitor and the enzyme; dissociation does not restore enzyme activity
•Sarin inhibition of cholinesterases
•Reversible inhibition involves equilibrium between inhibitor and enzyme; removal of inhibitor from the system will restore enzyme activity

Reversible inhibition can be categorized as competitive, noncompetitive and uncompetitive
•Competitive inhibitors competes with substrate for active site
•Noncompetitive inhibitors bind at a site distinct from active site (can occur with or without bound substrate)
•Uncompetitive inhibitors bind only to enzyme-substrate complex

Question 10

Describe enzyme classification, including 6 enzyme classes.

Answer 10

Nomenclature of measured enzymes standardized by Enzyme Commission (EC) of International Union of Biochemistry
•Categorizes enzyme by class  subclass  sub-subclass  enzyme number within sub-subclass
•Most enzymes are still referred to by their historical/practical names

Enzymes are generally classified into 1 of following 6 classes:
•Oxidoreductases: redox reactions
•Transferases: transfer of functional groups
•Hydrolases: hydrolysis reactions
•Lyases: group elimination to form double bonds
•Isomerases: isomerizations
•Ligases: bond formation coupled with ATP hydrolysis

Question 11

Define isozymes and provide an example.

Answer 11

Isozymes – enzymes that catalyze the same reaction but have different amino acid sequences
•Isozymes generally fold to similar tertiary structures thus conferring similar affinities for and catalytic rates with substrates
•Some isozymes have completely different protein structures (cytoplasmic vs mitochondrial forms of CK and AST)
•Isozyme nomenclature is based on electrophoretic migration
•Furthest migration from the anode is designated isozyme 1
•Examples of clinically relevant isozymes: LDH, CK, amylase, ALP

Question 12

Define isoforms and provide an example.

Answer 12

Isoforms – multiple forms of molecules (enzymes) due to post-translational modifications (PTMs)

• Multitude of PTMs can lead to many isoforms
• PTMs can be part of normal enzyme metabolism but can also arise pathologically
• PTMs will affect the physicochemical properties of the enzyme
• Affects catalytic activity and ability to detect analytically

Question 13

Define macroenzymes and provide examples.

Answer 13

Macroenzymes – high molecular weight protein complexes derived from cross-linking via immunoglobulins or spontaneous polymerization
•Characterized by type of molecular complexing
•Type I: immunoglobulin bound enzymes
•Type II: polymerization, binding to lipoproteins or drugs
•Macroenzymes undergo slower clearance due to size and will accumulate in the circulation
•Clinically benign but can cause diagnostic conundrums as macroenzymes still retain catalytic activity

Question 14

Provide strategies for macroenzymes.

Answer 14

Direct removal of macroenzyme
•PEG treatment
•Sepharose G resin (protein A)
•Ultrafiltration

Resolve macroenzyme from native enzyme
•Electrophoresis
•Size-exclusion chromatography

Measurement on multiple platforms

Urinary measurements

Question 15

Define enzyme kinetics.

Answer 15

Enzyme kinetics are fundamental to understanding catalytic activity
•Knowledge of enzyme kinetics allows for development of activators/inhibitors as therapeutics
•Manipulation of enzyme kinetics is the core of enzyme-based assays in the clinical laboratory
•Enzyme kinetic theory based on the two-step process of a catalyzed reaction

Question 16

Describe basics of enzyme kinetics.

Answer 16

Zero order reaction – rate of reaction is independent of substrate concentration
•Excess substrate relative to enzyme; all active sites are bound to substrate
•Reaction rate varies based on enzyme activity/concentration

First order reaction – rate of reaction is proportional and dependent on substrate concentration
•Enzyme concentration is constant; enzyme is in excess relative to substrate
•Reaction rate varies based on substrate concentration

Question 17

Define Michaelis Menten and provide the equation.

Answer 17

Michaelis and Menten developed a model by which enzymes catalyzed a reaction:
•Equilibrium is attained rapidly among enzyme, substrate and enzyme-substrate complex
•Formation of product is considered irreversible process
•Overall rate of reaction under otherwise constant conditions is proportional to concentration of enzyme-substrate complex

Michaelis-Menten equation describes the relationship between velocity of reaction to substrate concentration:
•Vmax is the maximum reaction velocity; reached when all enzyme is saturated with substrate
•[S] is the concentration of the substrate
•Km is the Michaelis or dissociation constant; equal to the substrate concentration at which reaction rate is at half of Vmax

Question 18

Draw MM and LB plots and describe how different modes of enzyme inhibition affect enzyme kinetics.

Answer 18

See slide 26.

Competitive inhibition – can be overcome by adding excess substrate relative to inhibitor
•Noncompetitive inhibition – cannot be overcome but does not affect enzyme affinity to substrate
•Uncompetitive inhibition – “combination” of both above modes of inhibition

Question 19

Provide an email of using enzyme kinetics in the clinical laboratory.

Answer 19

Creatine Kinase is an enzyme measured in the clinical laboratory, typically as a marker of muscle damage and/or inflammation (see slide 29)

Rate of reaction catalyzed in last step is directly proportional to the rate of product formation in the first step. This configuration allows for assessment of amount of creatine kinase present.

Enzymes are measured to assist in clinical medicine
•AST, ALT, GGT, ALP for liver disease
•CK for muscle disease
•Amylase for pancreatic disease
•Enzymes are heavily exploited for many spectrophotometric assays
•Cholesterol, Triglycerides, Creatinine

Question 20

How are enzymes reported?

Answer 20

Most – if not all – enzymes measured in the clinical laboratory are reported as unit of activity per volume (U/L or IU/L)
•Unit of activity refers to quantity of substrate consumed or product formed in a chosen unit of time
•(U) unit or (IU) international unit is defined as the quantity of enzyme that catalyzes 1 micromole of substrate per minute
•Recall: catalytic activity depends on many parameters (pH, aqueous environment, temperature, substrate, activators, etc.)
•These parameters are specified in the definition of (U) or (IU)

Question 21

How do we measure enzymes?

Answer 21

Simplistically, enzyme activity can be measured via appearance of product or disappearance of substrate.
•Unfortunately, products/substrates of enzymes of clinical interest are not as amenable for direct detection/measurement
•Common strategy is to utilize coupled reactions; generate an easily measurable analyte that is proportional to enzymatic activity
•Enzyme activity can also be assessed via measurement of reaction rates

Question 22

Define fixed-time and continuous monitoring reaction rate methods.

Answer 22

• Fixed-time methods: amount of change produced by the enzyme is measured after the reaction is stopped at the end of a fixed-time interval.
• Continuous-monitoring methods: progress of reaction is monitored continuously.

Question 23

Describe limitations of fixed time methods.

Answer 23

Fixed-time suffers from numerous disadvantages:
•Long incubation times
•Often an initial lag phase before zero order reaction achieved
•Reaction rate can fall off over time
•Reaction progress curve inevitably become non-linear over time
•Requires carefully attuned reaction conditions to ensure linearity within fixed time interval

Question 24

What are other methods to measure enzyme activity?

Answer 24

For the most part, enzyme activity is assessed via measurement of substrate spectrophotometrically (you will learn this later in the spectrophotometry lectures).
•Spectrophotometric assays can be end-point or kinetic-based methods

Other means to measure enzymes include:
•Enzyme mass concentration (renin)
•Immunoassay (reflect on limitations if enzyme naturally has many isoforms)
•Electrophoresis (isozyme differentiation)