CLINICAL CHEMISTRY CHAPTER 10

1
CLINICAL CHEMISTRYCHAPTER 10

• ENZYMES

2

• Introduction
• Enzymes are the chemical lubricants that make
  chemical reactions go nice and easy
• They help out with chemical reactions - but
  they dont get used up themselves ( sort of
  like the oil in your car hopefully )
• They are always proteins ! ( Remember proteins ?
  )
• Theyre found in cells throughout the body.
  When these cells get sick the enzymes in them
  tend to ooze out into the plasma
• Want to diagnose heart attacks, liver disease,
  muscle disease, pancreatitis, prostate cancer and
  bone disease? Enzymes can do that !
• They are different because we measure them not
  in terms of how many there are ( mg / dl , for
  example ) but in terms of their activity ( how
  well they are lubricating chemical reactions

3
KEY TERMS

• Activation energy
• Activators
• Apoenzyme
• Catalyst
• Coenzyme
• Cofactor
• Enzyme
• First Order Kinetics
• Second Order Kinetics
• Isoenzymes
• Holoenzyme
• International Unit (IU) of Enzyme Activity
• Kinetic Assay
• 2 - Point Assay

• Substrate
• Substrate Depletion
• Zymogen
• Prosthetic Group
• Inhibitors
• Competitive Inhibitors
• Non - Competitive Inhibitors
• Uncompetitive Inhibitors
• Allosteric Site
• Active Site
• Michaelis - Menten Constant
• Myocardial Infarction ( MI )
• Congestive Heart Failure ( CHF )

4
The Enzymes we will study

• CPK
• ALK
• AST
• ALT
• GGT
• LDH
• AMY
• LIP
• ACP
• SChE

5

• OBJECTIVES
• Discuss the general structure and functions of
  enzymes
• Define a unit of enzyme activity ( IU )
• Discuss how temperature, pH and other factors
  effect enzyme activity
• Explain first - order and zero - order enzyme
  kinetics and the special techniques that are
  used to measure enzymes
• Discuss the tissue sources, clinical
  significance and Normal Values of the following
• CPK, LDH, AST, ALT
• ALK, GGT
• AMYL, LIP
• ACID PHOS, Cholinesterase

6

• General Properties and Definitions
• Many chemical reactions cannot occur at 37 º (
  too cold )
• Many chemical reactions require added heat energy
• Biochemical reactions also require this energy ,
  but proteins will denature
• So how do biochemical reactions ever happen ?
  They cheat !!!
• Biochemical reactions can occur if there is a way
  to lower the amount of energy it takes to make
  the reaction happen.
• So how do biochemical reactions cheat ???

7

• Answer They get a catalyst
• Catalysts are substances that lessen the amount
  of energy required for chemical reactions to
  occur
• Catalysts are not used up in the reaction
• Substrate
  Product
• Enzymes are protein catalysts
• Enzymes are required for the numerous metabolic
  processes of all cells
• When cells are damaged, enzymes leak out into
  the plasma
• The measurement of these enzymes are useful
  diagnostic tools

Enzyme
8
Activation Energy for Chemical Reactions
Catalysts decrease the amount of energy required
to activate chemical reactions. Enzymes are
protein catalysts.
9

• Enzyme Structure
• Similar to protein structure, because all enzymes
  are proteins
• 1 º Amino acid sequence
• 2 º Interaction between 2 locations on the
  protein chain
• 3 º Folding of chains ( 3D structure )
• 4 º 2 or more separate polypeptide chains

10

• Definitions
• Active site
• Physical location on the enzyme molecule which
  interacts with the substrate molecule
• Allosteric Site
• Non active site , but which may interact with
  other substances to change the overall enzyme 3D
  shappe
• Isoenzymes
• Structurally different enzymes ( proteins ) but
  which catalyze the same chemical reactions

11

• Cofactor
• A non protein substance required for normal
  enzyme activity
• Cofactors maintain enzyme 3D structure ( critical
  for enzyme function )
• There are 2 types of cofactors
• Activators ( Inorganic --- Magnesium ,
  Calcium )
• Coenzyme ( Organic - NADH )

12

• Holoenzyme
• Enzyme coenzyme ( prosthetic group )
  Active enzyme
• Proenzyme ( Zymogen , Apoenzyme )
• Enzyme coenzyme Inactive enzyme

13
Enzyme Structure and Substrate
14

• Classification and nomenclature
• Enzyme kinetics
• Chemical reactions occur spontaneously if the
  energy for reactants is higher than products
• The amount of energy required to stimulate
  molecules to break their chemical bonds and form
  new bonds is the activation energy
• Increasing the temperature can generate the
  activation energy. This a problem for living
  cells

15

• Enzymes lower the activation energy for chemical
  reactions
• The enzyme substrate complex has a lower
  activation energy than the substrate alone
• Enzymes are usually specific as to which
  chemical reactions they catalyze

E S ES
E P
Enzyme Substrate Complex
Enzyme Substrate
Enzyme Product
The enzyme is not consumed or depleted by the
chemical reaction
16

• Enzymes differ in their ability to react with
  different substrates
• There are various enzyme specificities
• Absolute Catalyzes only 1 specific substrate
• Group Catalyzes reactions of a particular
  chemical group
• Bond Catalyzes reactions of particular
  chemical bonds
• Stereoisomerism Catalyzes reactions of
  steroisomers

17

• Factors that influence enzymatic factors
• First Order Kinetics
• Enzyme concentration exceeds substrate
  concentration
• As substrate concentration increases , the
  reaction rate increases
• The reaction rate is proportional to substrate
  concentration
• This is not a good place to measure enzyme
  activity ( The
  reaction rate is dependent on substrate , not
  enzyme )

18

• Zero Order Kinetics
• Substrate concentration is in excess
• Substrate saturates enzyme
• All enzyme reacts with the excess substrate
• The chemical reaction rate goes to maximum
  velocity
• Reaction rate is dependent on enzyme activity (
  conc . )
• Enzymes are not measured in terms of
  concentration , but activity
• Zero Order conditions are suited for enzyme
  measurement because this is where the reaction
  rate is dependent on the enzymes ability to
  catalyze a chemical reaction

19
Relationship between substrate concentration and
the reaction rate in a enzymatic reaction
Zero Order conditions
Reaction Rate
First Order Conditions
Substrate concentration
20

• Enzyme concentration
• Enzymes are not measured in terms of
  concentration
• Enzymes are measured is terms of their activity
• Enzyme activity The rate at which an enzyme
  catalyzes a chemical reaction
• Increased activity is proportional to increased
  concentration
• Activity is measured under Zero Order conditions
  because this is where the reaction rate is
  dependent on the work ( activity ) of the enzyme

21

• MICHAELIS-MENTEN CONSTANT (Km)
• Numerical constant for each enzyme and substrate
  under defined conditions
• Expresses a relationship between the reaction
  rate and substrate concentration

22

• pH ( acidity )
• Enzymes are proteins and subject to changes in 3D
  structure from pH changes
• pH must be carefully controlled in enzymatic
  reactions
• Temperature
• Reactions rates vary dramatically with
  temperature changes
• Reactions rates may double per 10 ? C increase
  in temperature
• Temperature must be defined and regulated for
  enzymatic reactions
• 37 ? C is the common standardized temperature

23

• Cofactors ( non-protein substances needed for
  enzymatic activity )
• Activators ( Ca2 , Fe 2 , Mg 2 , Mn 2
  , Zn 2 )
• Coenzymes ( Vitamins and nucleotide phosphates )
• Increased coenzyme concentration increases
  reaction rate

24

• Inhibitors ( Substances that decrease enzyme
  reaction rates )
• Competitive Inhibitors
• Substances that bind at the enzymes active site
• Competes with substrate for the active site
• Addition of additional substrate increases the
  reaction rate
• Non Competitive Inhibitors
• Substances that bind at an enzymes non active
  site
• Enzymes 3D shape is altered, decreasing enzyme
  activity
• Addition of additional substrate has no effect on
  reaction rate
• Uncompetitive Inhibitors
• Substances that bind with enzyme substrate
  complex

25

• Effects of inhibitors on Km
• Competitive Inhibitors increase Km
• Non-competitive Inhibitors have no effect on Km
  ( maximum
  velocity is not possible )
• Un-competitive Inhibitors decrease Km

26

• Measuring enzyme activity
• Enzymes are not directly measured
• Enzymes are commonly measured in terms of their
  catalytic activity
• We dont measure the molecule
• We measure how much work it performs
• Its catalytic activity
• The rate at which it catalyzes the conversion of
  substrate to product
• The enzymatic activity is a reflection of its
  concentration
• Activity is proportional to concentration

27

• Photometric measurement of activity
• E S ES
  E P
• Enzyme activity can be tested by measuring
• Increase of product
• Decrease of substrate
• Decrease of co-enzyme
• Increase of altered co-enzyme
• If substrate and co-enzyme are in excess
  concentration, the reaction rate is controlled by
  the enzyme activity.

28

• Coupled enzyme reactions
• Some enzyme testing utilizes other enzymatic
  reactions in a sequence of reactions.
• These other accessory enzymes are not being
  measured.
• Auxillary enzymes are used as reagents and
  added in excess so they do not become rate
  limiting factors in the overall process.
• All enzymatic reactions in a coupled assay must
  be performed at zero order kinetics.

29

• Example of a coupled assay
• S1 E1 S1 E1
  E1 P1 ( RXN 1)
• P1 E2 P1 E2
  E2 P2 ( RXN 2 )
• P2 E3 P2 E3
  E3 P3 ( RXN 3)
• E1 Enzyme we want to measure ( patient )
• E2 Auxiliary enzyme ( reagent )
• E3 Indicator enzyme ( reagent )
• There are 3 reactions with 3 different enzymes
• P1 becomes the substrate for reaction rxn 2
• P2 becomes the substrate for reaction rxn 3
• We end up measuring reaction 3, but its reaction
  rate is determined by reaction rate of rxn 1

30

• Enzymatic reactions are measured in terms of the
  rate at which substrate is converted into
  product. This can be done in 2 different ways.
• Fixed Time Assay ( 2 Point )
• Zero Order Kinetics
• Substrate concentration is measured at set timed
  intervals to determine enzyme activity
• A slight delay from the beginning of the reaction
  to maximum velocity is the lag phase
• These times intervals may not be short enough to
  detect sudden changes
• Extreme elevations in enzyme activity may deplete
  the substrate between measured intervals -
  Substrate Depletion
• Substrate depletion causes falsely decreased
  activity results.

31
Two Point Enzyme Methodology
32

• Multipoint Continuous Monitoring ( Kinetic Assay
  )
• Continuous measurements of substrate product
  concentration are recorded by the
  spectrophotometer of an automated analyzer
• Substrate depletion is detected because the
  analyzer is always looking and will see any
  sudden dramatic changes

33
Multipoint Enzyme Method
34
Comparison of 2 Point and Multipoint Enzyme
Techniques
35
Enzyme Activity can be measured as a function of
the change of absorbance values over a period of
time
Remember that enzymes are measured in terms of
activity How much work is getting done? How
fast are chemical reactions taking place? A
large change in absorbance per unit of time
indicates that the reaction was occurring at a
rapid rate, meaning that enzyme activity is high
36

• Calculation of enzyme activity
• Remember, its not concentration its activity
• Enzymes are measured in terms of how fast they
  convert substrate into product
• Activity is a measurement of how fast they work
• The common unit of enzyme activity is the
  International Unit ( IU )
• 1 IU That amount of enzyme that will convert 1
  micromole (1 ?mole ) of substrate to product
  per minute under defined conditions
• These conditions are such things as
• pH
• Temperature
• Substrate

37

• Mathematical expression of enzyme activity
• ?A The rate of change in Absorbance ( A )
  of the substance being measured
• MIN Time the reaction is measured
• TV Total sample volume ( patient plasma
  reagents )
• SV Volume of patient plasma
• 106 Conversion of moles into micromoles
• L Length of the light path
• ? Molar absorptivity

38

• Example of Molar Absorptivity calculation
• NADH ( a common co-enzyme ) absorbs light at 340
  NM
• NAD ( the reduced form ) does not absorb
  light at 340 nm
• Increased ( or decreased ) NADH concentration in
  a solution will cause the Absorbance ( A ) to
  change.
• A 0.05 X 10-3 Molar NADH solution placed in a 1
  cm light path has an absorbance of 0.311

The molar absorptivity of NADH is 6.22 x 103
Moles / Liter Each chemical substance has its
own unique molar absorptivity
39

• Enzymes as reagents
• Enzymes are used as reagents in a wide variety of
  methodologies to measure other non-enzyme
  substances
• Enzymes help to produce substances that can be
  easily measured
• Enzyme specificity helps to eliminate interfering
  substances
• If the enzyme is used as a reagent, we do not
  want the enzyme concentration to be a affect the
  chemical reaction
• To avoid this the enzyme is added in excess so
  that it cannot become a rate limiting factor .
  
• Remember, we are just using the enzyme to measure
  something else

40

• Creatine Kinase ( CK , CPK )
• CPK catalyzes
• Creatine ATP Creatine
  phosphate ADP
• High concentrations of CPK in muscle, cardiac and
  brain tissues
• Increased plasma CPK activity is associated with
  damage to these tissues
• ? CPK is especially useful to diagnose
• AMIs
• Skeletal muscle diseases ( Muscular Dystrophy )

CPK
41

• CPK ( cont )
• CPK has 3 isoenzymes
• Each isoenzyme is composed of two different parts
  ( dimers )
• CK - BB BRAIN
• CK - MB CARDIAC
• CK - MM SKELETAL MUSCLE
• Skeletal muscle CPK is 99 CK-MM
• Cardiac muscle CPK is 80 CK-MM and 20 CK-MB

42

• CPK ( cont )
• Most normal plasma CPK is CK-MM
• Because of CK MBs association with cardiac
  tissue, increased CPK MB ( gt 6 of the total
  CPK activity ) is a strong indication of AMI
• Post AMI CPK - MB
• CK-MB increases 4 8 hours post AMI
• Peaks at 12 - 24 hours post AMI
• Returns to normal 24 - 48 hours later

43

• CPK ( cont )
• CPK assays are often coupled assays CPK
• In the example below, the rate at which NADPH is
  produced is a function of CPK activity in the
  first reaction.
• Hexokinase and G6PD are auxiliary enzymes

Creatine Phosphate ADP
Creatine ATP

ATP Glucose
Glucose 6 - Phosphate
G 6 - P
NADP 6 -
Phosphogluconate NADPH
CPK
Hexokinase
G6PD
44

• CPK ( cont )
• RBCs lack CPK, but hemolyzed RBCs release
  adenylate kinase into the plasma, causing falsely
  increased CPK activity
• Hemolyzed specimens must be recollected
• Reference ranges
• Males Total CPK 15 - 160 IU / L
• Females Total CPK 15 - 130 IU / L
• Lets remember 15 150 IU / L
• CK - MB lt 6 of Total CPK

45

• Lactate dehydrogenase ( LDH , LD )
• LDH is found in many different tissues
• Skeletal muscle
• Cardiac muscle
• Renal tissue
• RBCs
• Plasma LDH activity is elevated in a variety of
  conditions
• Liver disease
• Cancers
• AMI
• Hemolytic diseases

46

• LDH ( cont )
• Lactate NAD
  Pyruvate NADH H
• At pH 9.0 the reactions moves to the right
• At pH 6.8 the reaction moves to the left
• NADH absorbs light at 340 nm NAD does not
• The reaction can be performed either direction

LD
47

• LDH ( cont )
• LDH post AMI
• Increases 12 24 hours post AMI
• Peaks at 48 72 hours
• May remain elevated for 10 days
• RBC hemolysis falsely increases plasma LDH
• Reference range 100 - 225 IU / L ( L ?
  P )

48

• LDH Isoenzymes
• Because increased total LDH is relatively
  non-specific, LDH isoenzymes can be useful
• 5 isoenzymes composed of a cardiac ( H ) and
  muscle ( M ) component
• LD - 1 ( HHHH ) Cardiac , RBCs
• LD - 2 ( HHHM )
• LD - 3 ( HHMM ) Lung, spleen, pancreas
• LD - 4 ( HMMM ) Hepatic and skeletal
• LD - 5 ( MMMM )
• A flipped LD - 1 / LD - 2 ( LD-1 gt LD-2 )
  consistent with AMI

49

• Aspartate Aminotransferase ( AST, SGOT, GOT )
• High concentrations of AST are found in
• Skeletal muscle
• Cardiac muscle
• Liver tissue
• Lung tissue
• Post AMI
• Rises 6 8 hours
• Peaks at 24 hours
• Returns to normal by day 5

50

• AST ( cont )
• AST assays usually utilize a coupled enzyme
  technique
• Aspartate ? - Keto - Glutarate
  Oxaloacetate Glutamate
  
• 
  
• Oxaloacetate NADH H
  Malate NAD
• MD is an auxiliary enzyme
• NADH absorbs light at 340 nm NAD does not
• Reference Range 5 - 30 IU / L

AST
MD
51

• Alanine aminotransferase ( ALT, SGPT )
• Highest concentrations in the liver
• Increased plasma ALT closely associated with
  liver disease

Alanine ?- Ketoglutarate
Pyruvate Glutamate Pyruvate NADH
H Lactate
NAD NADH absorbs light at 340 nm NAD
does not Reference range 5 - 35 IU / L
ALT
LD
52
Chemical reactions of the amino-transferases AST
and ALT
53

• Alkaline phosphatase ( ALK, ALK PHOS )
• Optimal pH at 9.0 10.0 ( alkaline )
• Removes phosphates ( PO4 ) from organic compounds
• Requires Mg as an activator
• High concentrations in
• Bone
• Liver
• Best utilized to diagnose bone and liver disease
• Reference range 30 90 IU / L

54

• Amylase ( AMY, AMYL )
• Catalyzes breakdown of starch and glycogen to
  glucose
• High concentrations in
• Pancreas
• Saliva Chew on some bread !!!
• Amylase is filtered into the urine ( unusual for
  a protein )
• Urine amylases are occasionally ordered
• Increased plasma or urine amylase is very
  suggestive of pancreatitis or pancreatic
  malignancy

55

• Amylase ( cont )
• Because amylase is concentrated and excreted in
  the urine, urine amylase values may be more
  diagnostic that plasma
• Many different amylase methodologies varying in
  the types of substrates used
• Reference range 60 180 SU / dl

56

• Acid phosphatase ( ACP, Acid Phos )
• Same function as ALK PHOS, but at a different pH
  
• Optimal pH is 5.0 ( acid )
• High concentrations in the prostate gland made it
  a useful test for the diagnosis of prostate
  cancer
• Its use is declining because of other, better
  tests for prostate cancer ( Prostate Specific
  Antigen - PSA )

57

• Gamma Glutamyltransferase ( GGT )
• High concentrations in liver tissue
• Increased plasma GGT is associated with
• Hepatobiliary disease
• Alcoholic cirrhosis
• Insurance companies utilize GGT to detect
  alcoholism
• Reference range 6 45 IU / L

GGT Glutathione amino acid
Glutamyl peptide L-Cysteine
58

• Lipase ( LIP )
• Hydrolyze ( breaks down ) fat
• High concentrations in the pancreas .
• Increased plasma lipase associated with
  pancreatitis
• Lipase is more specific than amylase for the
  detection of pancreatitis
• Reference range 0.0 - 1.0 U / ml

59

• Pseudocholinesterase ( SChE )
• Considered a screening test for exposure to
  organophosphate exposure ( pesticides)
• SChE is found in plasma, liver, pancreas and
  brain
• Reference Range 4,000 - 12000 IU / L

60

• Diagnosis of AMI ( Acute Myocardial Infarction
  )
• Remember cardiac disease is the 1 cause of
  death in the United States
• 1 Symptoms, physical examination , and patient
  history But there are many
  causes of chest pain other than AMI
• 2 EKG only 50 reliable - may be normal
  even during AMI
• 3 Laboratory tests ( Troponin I , CKMB ,
  Myoglobin , BNP )
• Cardiac cellular necrosis following ischemic
  event causes soluble proteins to leak into the
  plasma
• Rephrased When coronary arteries are blocked
  ( ischemic event ) cardiac tissue is deprived of
  oxygen and dies, and dead cardiac tissue cells
  spill their guts into the plasma

61

• Myoglobin
• Soluble heme protein
• Present in all muscle cells, cardiac and
  skeletal
• Plasma myoglobin is elevated in various forms of
  muscle damage - surgery, strenuous exercise,
  degenerative muscle diseases and physical trauma
• Myoglobin can also be elevated from decreased
  renal clearance
• Relatively low molecular weight , water
  solubility and high cellular cytoplasm
  concentrations cause myoglobin to be the first
  marker to be released from damaged cells

62

• Troponin I
• Contractile protein associated with cardiac and
  skeletal tissue
• Two forms Troponin I and Troponin T
• Troponin I from cardiac tissue has a unique
  antigenic structure that differentiates it from
  skeletal Troponin I and facilitates its
  measurement
• Of all the cardiac markers , Troponin I is the
  most specific for cardiac injury
• CK-MB
• Iso-enzyme found in cardiac and skeletal tissue
• Plasma CKMB concentrations are increased in AMI
  and various forms of skeletal muscle trauma
• Although not absolutely specific, very high
  concentrations in cardiac tissue make it a good
  marker for AMI

63

• B- Type Natremic Protein ( BNP )
• Increased plasma BNP is associated with
  Congestive Heart Failure ( CHF )
• CHF is one of the most common reasons for
  hospitalization in patients greater than 70 years
  old
• BNP assays are often ordered with AMI markers to
  differentiate between AMIs and CHF
• BNP in normal in AMI

64

• Cardiac enzymes
• In the good old days , AMIs were diagnosed with
  the cardiac enzymes
• The cardiac enzymes consisted of Total CPK,
  LDH and AST
• Although still useful, the more specific markers
  such as Troponin I, Myoglobin and CK-MB have
  largely replaced the cardiac enzymes

65
TRIAGE CARDIAC PANEL FOR RAPID QUANTIFICATION
OF CK-MB, MYOGLOBIN , TROPONIN I and
BNP

• TRIAGE is a common commercial test package that
  quantitates the three most significant laboratory
  markers for AMI - CK-MB , Troponin I and
  Myoglobin
• Procedure summary
• Lithium heparin whole blood is added to the
  cartridge system
• Plasma separates from RBCS after passing through
  a filter
• Plasma ( CK-MB , Troponin I and Myoglobin )
  reacts with fluorescent tagged anti-CKMB,
  anti-Troponin and anti-Myoglobin monoclonal
  antibodies in the reaction chamber
• Concentration of the analyte is directly
  proportional to fluorescence that is measured on
  the Triage Meter

66

• Triage system cut - off ranges for cardiac
  markers . Values above the cut-off ranges
  indicate possible AMI
• Troponin I lt 0.6 ng / ml
• Myoglobin lt 107 ng / ml
• CK-MB lt 4.3 ng / ml
• Reference ranges for the cardiac enzymes
• Total CPK 15 - 150 U / L
• LDH 100 - 225 U / L
• AST 5 - 30 U / L

67
Summary of Triage cardiac markers after AMI
The interpretation of cardiac markers can be
difficult because blood specimens are not always
collected soon after patients suffer chest pains
- delays of hours to days are common before
patients seek medical attention
The most common symptom of a heart attack is
.
Although any single analyte may not be adequate
to diagnose an AMI, the collective information
from all three analytes can be very useful
68
Enzyme Top 10

• Enzymes are protein catalysts that lower
  activation energy of chemical reactions
• Enzymes are not consumed in the reactions they
  catalyze
• Enzymes are measured in terms of activity ( know
  the definition of an IU )
• Enzymes are measured at zero order kinetics
• CPK ? Cardiac and skeletal muscle
• ALK ? Bone and hepatic tissue
• GGT and ALP ? Hepatic tissue
• AMY and LIP ? Pancreas
• LDH ? Hepatic, Cardiac ,
  RBCs ( lots of others )
• AST ? Cardiac, Hepatic and skeletal
• CK-MB ? Cardiac ( good to
  diagnose AMI )

69

• REFERENCE RANGES
• ALT 5 - 35 IU/L
• AST 5 - 30 IU/L
• CPK 15 - 150 IU/L
• GGT 5 - 45 IU/L
• LDH 100 - 225 IU/L
• ALK 30 - 90 IU/L
• AMYL 60 - 180 SU/Dl
• LIPASE 0.0 - 1.0 IU/ML
• CK-MB LESS THAN 6 OF CPK
• ACID PHOS 3.0 - 12.0 IU/L
• MYOGLOBIN 30 90 mg/dl
• TROPONIN I lt 0.6 ng/ml
• BNP lt 100 pg/ml

70
Enzyme Links
http//www.bact.wisc.edu/microtextbook/Metabolism/
Enzymes.html http//med6.bham.ac.uk/teaching/clin
year3/enzymslide/ http//ull.chemistry.uakron.edu
/genobc/Chapter_20/ http//users.rcn.com/jkimball
.ma.ultranet/BiologyPages/E/Enzymes.html