Part 1 Enzymes - Study Guide

What are enzymes?

• biological catalysts that speed up chemical reactions involving formation/ breaking of a covalent bond

• they lower the activation energy of the reaction by stabilizing the transition state

• they do NOT change the equilibrium constant or the standard free energy

What is the reaction model for enzymes?

E + S —> ES → E + P

• enzyme binds substrate → forms complex → releases product

What is an active site?

• pocket on the enzyme where the substrate binds

  • lock and key: substrate fits the rigid active site

  • induced fit: active site changes shape after substrate binding to optimize the catalyst

How are enzymes catalyzed?

• based on the type of reaction catalyzed

• most enzymes end in -ase

What type of enzyme involves the transfer of electrons (oxidation/reduction)

• Oxidoreductases

  • oxidases

  • dehydrogenases (LDH)

What enzyme involves the transfer of a chemical group from one molecule to another?

• Transferases

  • transaminases (AST/ALT)

  • transmethy;ases, transacetylase, kinases

What enzyme involves cleavage by addition of water?

• Hydrolases

  • esterases

  • proteases

  • amylase

  • lipase

  • phosphatases (ALP/ACP)

What enzyme involves splitting or forming C-C, C-O, or C-N bonds without hydrolysis or oxidation?

• Lyases (desmolases)

What enzyme involves rearrangement of atoms within a molecule (changing geometry/shape)?

• Isomerases

What enzyme involves joining two molecules using energy from ATP (or another triphosphate) hydrolysis?

• Ligases

What happens during first-order?

• at LOW S (substrate concentration) v (velocity change) is directly proprtional to S

• doubling S, doubles the rate

What happens during Mixed Order?

• at intermediate (S), v is not simply proportional to S

What happens during zero-order?

• at very HIGH (S), v reaches Vmax and is independent of (S)

• all enzyme active sites are saturated

• this region is used for chemical assays

What is the Michaelis-Menten equation?

• v = (Vmax x S) / (Km + S)

  • Vmax = the maximum velocity reached when the enzyme is fully saturated with substrate

  • Km = Michaelis constant, substrate concentration at which v = ½ Vmax

    • reflects how tightly the enzyme binds substrate

    • LOW Km = high affinity

  • when S is much greater than Km, v = Vmax (zero - order kinetics)

Why is the Lineweaver-Burk plot used?

• taking the reciprocal of the Michaelis-Menten equation gives a straight line, which makes Km and Vmax easier to determine

What is the Lineweaver-Burk equation?

1/v = (Km / Vmax) ( 1/S) + 1/Vmax

• plot 1/v on y axis vs 1/S on the x axis

• slope = Km / Vmax

• y intercept = 1/Vmax

• x intercept = -1/Km

Why do clinical assays use zero - order kinetics

• we want to measure the amount of enzyme present, NOT how much substrate is around

What does zero-order mean?

• S (substrate concentration) is no longer rate-limited, so the only thing that changes the reaction rate is the enzyme concentration itself

• to ensure this, substrate concentration is set at 10-100 x km

  • ensures all enzyme is saturated regardless of patients enzyme level

What factors affect enzyme activity?

• enzyme concentration

• substrate concentration

• temperature

• pH

• cofactors / activators

• inhibitors

• time

How does enzyme concentration affect enzyme activity?

• rate is directly proportional to (E) when (S) is in excess

• this is what zero-order assays measure

How does substrate concentration affect enzyme activity?

• rate rises with (S) until saturation is at Vmax

  • must keep S > 10-100 x Km for valid assays

How does temperature affect enzyme activity?

• rate doubles for every 10 C increase

• denaturation occurs at 40 - 60 C

What is the optimal temperature for running enzyme assays?

• 37 C (body temp)

  • lab assays run at constant 30 - 37 C

How does pH affect enzyme activity?

• optimal pH at 7-8

• extreme pH alters ionized groups / denatures enzymes

• must use proper buffer

How do cofactors/activators affect enzyme activity?

• required for activity of many enzymes

  • ex: Mg2+ for CK and ALP

  • Cl- for amylase

Why are cofactors clinically relevant?

• anticoagulants like EDTA and citrate bind metal cofactors and falsley lower activity

  • ex: use heparin for ALP

How do inhibitors affect enzyme activity?

• reduce activity by binding the enzyme

• can be competitive, noncompetitive, or mixed

How does time affect enzyme activity?

• reaction progresses until substrate runs out or product builds up

  • kinetic assays must read during the zero-order phase

T/F: temperature MUST be held constant during an assay and MUST be identical from run to run?

• True

What can happen to enzyme assays during low temperatures?

• lower temps do NOT denature enzymes, repeated freezing/thawing damages them

What temps can enzymes be stored at?

• 4 C

• -20 C

• -80 C

What enzyme can be stable at room tmp for 24 hrs?

• amylase

What enzyme has activity that INCREASES after freezing/thawing or lyphilization

• ALP

What temperatures can LDH be kept at?

• stable at RT

• LD-4 and LD-5 : inactivated at 2-8 C

define cofactor.

• non-protein helper required for enzyme activity

• can be organic (coenzyme) or inorganic (metal ion)

define coenzyme.

• organic, non-protein cofactor that is loosely bound and often shuttles between enzymes

  • ex:

    • NAD+, NADP+, biotin

    • thiamine pyrophosphate, pyroxal phosphate, coenzyme A

Define prosthetic group.

• coenzyme that is tightly (often covalently) bound to the enzyme

Define Holoenzyme.

• enzyme + prosthetic group (fully active form)

Define Apoenzyme.

• enzyme without cofactor (inactive form)

  • ex: zinc deficiency, aldolase because it lacks Zn2+ cofacto

Define metal ion activators.

• inorganic cofactors

  • Cl- for amylase

  • Mg2+ for ALP and CK

  • Zn2+ for aldolase and other enzymes

  • Mn2+, Fe2+, Ca2+, K+

What are Competitive Inhibitors?

• resembles the substrate and competes with the active site

• increases Km (lower apparent affinity), but Vmax is unchanged

  • you can overcome it with more substrate

What are Noncompetitive Inhibitors?

• bind at a site other than the active site

• decreases Vmax, Km is unchanged

• adding substrate does NOT overcome it

What is a competitive/Mixed Inhibitor?

• bind the ES complex or both E and ES with different affinities (mixed)

• both Vmax and Km decrease (uncompetitive) or change (mixed)

What do enzyme assays measure?

• plasma enzyme amount are too low to detect by direct mass measurement

• measures enzyme activity

  • how fast the enzyme converts substrate into product

What does a reaction-rate (kinetic) assay measure?

• enzyme activity

What does a end-point (one-point) assay measure

• enzyme activity OR concentration depending on conditions

How does the Kinetic Assay work?

• absorbance is read repeatedly throughout the incubation

• advantages

  • can verify zero-order conditions

  • exclude lag phase

  • can detect substrate exhaustion

How does an endpoint assay work?

• absorbance is read once at the end of a fixed incubation period

• Abs/min is then calculated

• allows batch processing of many samples at once

what reaction is used in many enzyme assays?

• many are coupled to a reaction involving NAD+ —> NADH or NADP —> NADPH

What absorbs strongly at 340 nm?

• reduced form (NADH or NADPH)

What does NOT absorb at 340 nm?

oxidized form NAD+ or NADP+

What does it mean if there is an increase in absorbance at 340 nm?

• NADH is being produced

  • forward LDH reaction

  • forward ALP reactions in some assays

What does it mean if there is a decrease in absorbance at 340 nm?

• NADH is being consumed

  • AST and ALT coupled assays

  • reverse LDH

What is 1 IU?

• the amount of enzyme that catalyzes the conversion of 1 umol of substrate to product per minute under defined assay conditions

• expresses activity NOT mass

What is important to know about the International Unit IU?

1 mU/mL = 1 IU/L

What is hemolysis?

• rupture of red blood cells releasing their contents into serum/plasma

How does hemolysis falsely increase enzyme results?

• Hemoglobin is colored (absorbs at 410 nm), which spectrophotometrically interferes with assays

• RBC’s contain most of the same enzymes being measured

How does hemolysis and hemoglobin affect lipase?

• it inhibits it so it falsley decreases results

What enzymes have their primary tissue source as the liver?

• Alanine aminotransferase (ALT,SGPT) - most hepatospecific

• Gamma-glutamyl transferase (GGT)

What enzyme has their primary tissue source in the pancreas?

• Amylase - and salivary glands

• Lipase - and some in stomach leukoytes and adipose

What enzyme has their primary tissue source in the prostate?

• Acid phosphatase (ACP)

  • and bone osteoclasts and RBC

What enzyme has their primary tissue source in the liver, heart, and erythrocytes?

• Lactate dehydrogenase (LDH)

What enzyme has their primary tissue source in the liver, heart, and skeletal muscle

• Aspartate aminotransferase (AST, SGOT)

What enzyme has their primary tissue source in the heart, skeletal muscle, brain?

• Creatine kinase (CK)

What enzyme has their primary tissue source in the liver, bones, intestines, and placenta?

• Alkaline phosphatase (ALP)

What enzyme has their primary tissue source in the liver, brain, and RBC?

• Cholinesterase (CHE)

What enzyme is tetrameric and has four subunits combined from two types: H (heart) and M (muscle/liver)

• Lactate dehydrogenase

What are the 5 isomers of Lactate Dehydrogenase?

• LD-1: HHHH

• LD-2 HHHM

• LD-3 HHMM

• LD-4 HMMM

• LD-5 MMMM

T/F: LD-1 migrates fastest to the anode in electrophoresis and LD-5 travels the slowest.

• True

What is the normal pattern for Lactate Dehydrogenase

• LD-2> LD-1

What is the “flipped LD ratio”

• LD-1 > LD-2

  • indicates:

    • acute MI

    • hemolysis

    • renal infarction

What does it mean if LD-3 is elevated?

• lung injury

What does it mean if LD-4/LD-5 are increased

• liver and skeletal muscle injury

  • LD-5 alone is more suggestive of liver disease

What is the reaction for Lactate dehydrogenase (LDH)?

• lactate + NAD+ → pyruvate + NADH + H+

• measures absorbance of NADH at 340 nm

What is a major source of error for Lactate Dehydrogenase?

• Hemolysis

  • RBC containing 100 - 150x more LDH

• Store at room temp

  • LD4 and LD5 are destroyed by refrigeration.