Environmental and Natural

Products Biochemistry
Biology 439
Dr. Bruce Greenberg

B2-154 ext.33209
greenber@uwaterloo.ca
 Lecture: T/T 1:30 PM - 2:20 PM – DC 1351
Tutorial (optional): M 10:30 AM - 11:20 AM – DC 1351
Dr. Bruce M. Greenberg, Biology Bldg. 2, Rm 154, x33209
e-mail: greenber@uwaterloo.ca
Office hours: T/T, 11:30 AM -12:20 PM, or by appointment
Course Website: https://learn.uwaterloo.ca/
Description
This course deals with the functions, distribution and environmental action of
natural compounds produced by plants and other biological systems. The
pathways of primary metabolism emphasized in general biochemistry courses will
not be covered. Instead, the emphasis will be on those compounds usually
described as secondary metabolites, i.e. those apparently “non-essential” products
whose physiological and ecological functions are either obscure or are of
peripheral importance to the organism. However, many of these “non-essential”
products have profound competitive, environmental, economic and
pharmacological significance; and as research proceeds, their physiological roles
within the parent organisms are becoming clearer. As well, this course has a
strong emphasis on how chemical and physical processes in the environment
impact on living organisms and their biochemistry.

Prerequisites
At least one-term course in organic chemistry and one-term course in
biochemistry.
Course Content

Introduction: Products of primary and secondary metabolism. Importance of

secondary metabolites. Review of chemical of isomerism, and
classification of enzymes.
Cytochromes and porphyrins: Overview of cytochromes and the roles of
cytochrome P450.
Isoprenoids: Mevalonate pathway. Terpenes, carotenoids, steroids, and
polyisoprenes.
Allelopathic Chemicals: Compounds that provide a competitive advantage
to an organism.
Phenolics: The shikimic acid pathway with an emphasis on the flavonoids.
Taxol: An important pharmaceutical chemical produced by a plant.
Environmental Photochemistry & Photobiology: Phototoxic compounds,
photodynamic therapy and photochemistry of beer.
Other topics
Alkaloids: Several diverse groups of nitrogen containing compounds.
Polyketides: Coupled C2 units, A key route for production of secondary
metabolites in bacteria and fungi.
Polyacetylenes, Amines, and organic cyanides: Potential antibiotics and
poisons.
Support Material: There is not a suitable text for this course. There is a
set of papers that is required for the course. Some of the papers
are background on different subjects covered, others are
examples of current research involving environmental and natural
products biochemistry. The material is assigned on a weekly
basis, as indicated in the table of contents for the papers. The
papers are posted to the course website.

1 1. Natural plant chemicals: Sources of industrial and medicinal materials

M.F. Balandrin, J.A. Klocke, E.S. Wurtle, W.H. Bollinger
1 2. Evolution of secondary metabolites from an ecological and molecular
phylogenetic perspective (Pages 1-6; rest of article optional)
M. Wink
2 3. Allelopathy – a natural alternative for weed control
F.A. Macıas, J.M.G. Molinillo, R.M. Varela, J.C.G. Galindo
2 4. Common and uncommon cytochrome P450 reactions related to
metabolism and chemical toxicity
F.P. Guengerich
3 5. Enzymatic hydroxylation reactions
H.L. Holand, H.K. Webber
3 6. Crawling Out of the RNA World
T.R. Cech
3 6a. Optional: Specific interaction between the self-splicing RNA of Tetrahymena
and its guanosine substrate: implications for biological catalysis by RNA
B.L. Bass, T.R. Cech
4 7. Serving up science for everyday cooks and gourmets alike
D.M. Schwartz
4 8. Biosynthesis of cyclic carotenoids
G. Sandmann
5 9. Evolution of floral scent in Clarkia: Novel patterns of S-Linalool synthase gene
expression in the C. breweri flower
N. Dudareva, L. Clarke, V.M. Blanc, E. Pichersky
5 10. The diversity of naturally produced organohalogens
G.W. Gribble
6 11. Flavonoid biosynthesis: A colorful model for genetic, biochemistry, cel biology,
and bitechnology
B. Winkel-Shirley
6 12. Identification of the flavonoid glycosides that accumulate in Brassica napus L.
cv. Topas specifically in response to ultraviolet B radiation
K.E. Wilson, M.I. Wilson, B.M. Greenberg
 7 13. The shape of things to come: Structural and synthetic studies of taxol and
related compounds
D.G.I. Kingston
7 14. Taxol and taxene production by Taxomyces andreanae, and endophytic
fungus of pacific yew
A. Stierle, G. Strobel, D. Stierle
8 15. Environmental Phototoxicity
R.A. Larson, M.R. Berenbaum
8 16. Toxicity of a PAH photooxidation product to the bacteria Photobacterium
phosphoreum and the duckweed Lemna gibba: Effects of phenanthrene and
its primary photoproduct phenanthrene quinone
B.J. McConkey, C.L. Duxbury, D.G. Dixon, B.M. Greenberg
9 17. Photodynamic therapeutics: Basic principles and clinical applications
W.M. Sharman, C.M. Allen and J.E. van Lier
9 18. Current Clinical and Preclinical Photosensitizers for Use in Photodynamic
Therapy
M.R. Detty, S.L. Gibson, S.J. Wagner
10 19. Interplay of photochemistry and beer: How lightstruck flavor is formed and
how it can be prevented.
D. De Keukeleire
11 20. Alkaloid biosynthesis - The basis for metabolic engineering of medicinal
plants.
T.M Kutchen
 Marking:
1st Midterm Exam Thur. Oct 31, 2019 50%
(Location TBA)
2nd Midterm Exam During final exam period 50%
(The 2nd Midterm Exam will only cover material from the
second half of the course)

Extra Credit: A 5 page (double spaced) paper on any subject

relevant to the course. Choice of topic does NOT need my
approval. NO MORE THAN 5 PAGES. The 5 pages are for
the body of the text. Figures, tables and bibliography are
extra pages. At least 2 entries in the bibliography must be
from the traditional, peer reviewed literature (i.e., a
published book or refereed journal).
Due: Thur. Nov. 21st, 2019 End of Lecture 10%
Schedule This Fall
Tue Oct 1: No class (Rosh Hashannah)
 1

1. Introduction
• A survey course dealing with the chemistry,
function, environmental effects and
distribution of natural products from plants
and other organisms.
• Disciplines involved: biochemistry, plant
physiology, plant molecular biology,
ecology, microbiology, entomology,
pharmacology, toxicology and food
science.
 2
What kind of products will be
involved in this course?
• Secondary metabolites (2°) not primary
metabolites (1°) will be emphasized.
• 2°metabolites; does not mean unimportant.
• Produced along offshoots of primary pathways.
• Often induced by varying environmental
conditions.
• Many have significant ecological, economic
and pharmacological uses.
 3

• Dividing line between 1°and 2°metabolism

not clear.
• Both processes are closely connected.
Primary metabolism provides the chemical
building block for all important 2°
metabolites.

Three major biosynthetic pathways are the

starting points for many 2°metabolites:
a. Shikimic Acid Pathway
b. Mevalonate (Isoprenoid) Pathway
c. Acetate-Malonate Pathway
 Photosynthesis
4

Carbohydrates
Pentose- P
Erythrose-4-P
Shunt
glycolysis

Phosphoenol Pyruvate Shikimic Acid

NH3
Acetate-malonate Pyruvate
pathway Amino Acids Aromatics
Acetyl CoA phenolics
lignans
Polyketides flavonoids
TCA Cycle
mevalonate
Polyphenolics
Porphyrins

Fatty Acids Alkaloids

caffeine
polyacetylenes Proteins cocaine
thiophenes
Nucleic Acids ephedrine
prostaglandins
ergotamine
Amines morphine
nicotine
Isoprenoids
Polyamines serotonin
terpenes
steroids
carotenoids Organic Cyanides
cyanogenic glycosides
 5

Three criteria help distinguish 2°

metabolites
a. Restricted distribution, mainly in plants and
microorganisms. Generally characteristic of
particular families, genera or species.
b. Formed along special biosynthetic
pathways.
c. Usually considered to be marginal or non-
essential to primary life processes.
 6
Some of the biological functions of
2° metabolites
a. Attract insect and bird pollinators to flowers.
b. Repel predators by smell, taste, toxicity.
c. Act as phytoalexins (antibiotics) in plants.
d. Reduced competition from other plant species,
i.e. allelopathy.
e. Formed to detoxify potentially harmful
compounds.
f. Storage products that can be catabolized for
energy and for simpler metabolites.
 7
Examples of natural products used in
medicine, agriculture and industry

a.Vitamin E (α-tocopherol), β-carotene

(precursor of Vitamin A) and ergosterol
(precursor of Vitamin D) are all products of
the isoprenoid pathway.
High dosages of the first two often recommended
for their antioxidant activity, i.e. they are effective
scavengers and quenchers of reactive oxygen.
 8

b. Insecticides, e.g. nicotine (alkaloid), rotenone

(isoprenoid and SAP pathways), pyrethrins
(SAP, polyketide, isoprenoid pathway).
c. Waxes and oils for paints and cosmetics. A
variety of sources.
d. Dyes for foods and cloth. Red, orange, blue,
purple, yellow and brown from many sources.
e. Spices and flavors, e.g. vanillin, peppermint,
orange and lemon, clove, cinnamon.
f. Perfumes (Largely isoprenoids).
 9
g. Pharmaceuticals:
• Cascara sagrada and senna extracts
(anthroquinones, laxatives).
• Castor oil (polyketide, laxative).
• Steroids (isoprenoids).
• Codeine (alkaloid, analgesic).
• Morphine (alkaloid, narcotic).
• Digitoxin (cardiac glycoside, heart disease).
• Ephedrine (alkaloid, bronchodilator).
• Taxol (mixed synthesis, anticancer drug).
Many plant products are poisonous, and even 10
familiar foods and drinks may contain
substances that can cause illness or may even
be carcinogenic

a. Chamomile tea can cause anaphylactic shock or

allergic rhinitis in sensitive individuals.
b. Apples may contain relatively high levels of
formaldehyde.
c. Broccoli contains a compound not unlike dioxin.
d. Peanuts often contaminated with a fungus that is
carcinogenic.
e. Celery contains psoralens which become reactive and
even carcinogenic when exposed to light.
f. Potato peels may contain high levels of solanine
which blocks cholinesterase, i.e. just as the
organophosphate insecticides.
 11
Many animals, including humans, have developed
metabolic defenses that protect us against
moderate doses of these substances.

However, the toxins that plants produce as

protection against bacteria, fungi and insects can
be present in amounts that are dangerous to
humans.

For example, environmental stress or attacks by

organisms can trigger large increases in the
production of these natural pesticides.
 12
Fortunately, many fruits and vegetables also
produce compounds that are natural cancer
inhibitors. Thus we can visualize a balance in
our natural food supply between cancer-
causing and cancer inhibiting substances.

Must be cautious in efforts to bio-engineer

disease-resistant and insect-resistant varieties.
A potato variety developed with high resistance
to insect attack had to be withdrawn from the
market- it was too toxic for human consumption!
 13
Obviously, there is tremendous promise in
studies of natural product chemistry and
associated topics.

More than 16,000 natural products have been

identified from plants, yet only 5-10% of all
land-plant species have been screened, and
the oceans remain an untapped source of
natural chemicals.

Environmental change also induces different

level of 2° metabolism.
 I1
2. Review of Chemical Isomerism
• Different molecules with the same chemical
formula
• Isomerism very important in natural product
and environmental biochemistry – many
natural compounds have complex structures
with many chiral centers
• Examples:
– Assume a bottle labelled: 46 % C2H6O
• CH3CH2OH or CH3OCH3
 I2
Examples continued
• Enzymes add groups stereochemically
– e.g., Steroid hydroxylation
OH

HO HO

• Enzymes only recognize one form of a structural or

stereoisomer
– e.g., Only L-amino acids are used in biology
• Aspartame (D-Asp-L-Phe)
• Sweet flavor
• Not recognized by enzymes, no calories
 I3
Structural Isomers
• Same molecular formula with different
arrangement of atoms and different physical
properties
• Not just mirror images
• Alkanes, alkenes and alkynes < 4 C do not
have isomers
– CH4, CH3CH3 and CH3CH2CH3
– Functional groups (=O, OH, Cl, etc.) can alter
this requirement
• Atoms must be ordered (arranged) differently
in space with respect to each other
 I4

Structural Isomers
• Must be a different configuration, not just a
different conformation
Conformers

Isomers
 I5
Structural Isomers

CH3 CH3 CH3

H C CH3 CH3 CH2 CH2 CH3 CH2 CHOH

CH3 CH2OH CH3
2-methylpropane n-butane 1-propanol 2-propanol

CH3(CH2)5 C CH
CH3
one triple bond
CH3
O
CH2OH
CH3 two rings
CH2CH3
ethanol
dimethyl ether
one double bond
+one ring
 Another example of structural I6

isomers
• Linear vs. cyclic sugars
O

HO O OH
OH HO
HO
HO OH
OH
O
H
OH

D-Glucose b-D-Glucose
 I7
Stereo isomerism
• Stereo isomers: Same bond order, different
arrangement of atoms in space
• Same chemical formula if spatial arrangement not
indicated
• For example: CHCl=CHCl could be
Cl H Cl Cl
or
H Cl H H
• Can be geometric or optical isomers.
• Geometric: different physical properties
• Optical: same physical properties, except rotation
of polarized light
 Stereo isomerism - Geometric isomers I8

Cis-trans arrangments – Fn groups are rigidly

fixed in space – Alkenes and ring compounds
H
H Cl H CH3 H

H COOH
C C C C
Cl CH3 Cl Cl COOH H

trans-1,2-dichloro- cis-1,2-dichloro- cis-(Z)-cinnamic trans-(E)-cinnamic

propene propene acid acid

Cahn, Ingold, & Prelog rules

CH3 F CH3 I
Cl
Atomic#: 6 7 8 9 17 35 53
C C C C
I Br Cl I I Br F C N O F Cl Br I
Increasing priority
(Z) (E)
 CH3 CH3 I9
CH3

cis – 1,3-dimethylcyclohexane CH3

trans – 1,3-dimethylcyclohexane
CH3
CH3
CH3
cis
CH3 trans

H H

trans
junction
H trans - decalin H
H

H
cis junction
H cis - decalin
H
 Optical Isomers I10

• Have one or more chiral centers – assymetric carbons

• Enantiomers - Stereo isomers that are Not Super-
imposable Mirror Images (NSMI)
• Opitcally active – rotate plane polarized light
• Enantiomers have identical physical properties – MP, BP
Density, Absorption spectrum – Except they rotate
polarized light in opposite directions
• Same amount of rotation, just opposite directions
• Equal amounts of enantiomers in a mixture
– Racemic mixture
– Optically inactive (net rotation is zero)
• Enantiomers have identical chemical reactivities, except
in the presence of another stereoisomer – such as an
enzyme!!
 I11

COOH COOH
Enantiomers of C
C H H OH
HO lactic acid (NSMI)
CH3 CH3

(-) L (+) D
S R
+ & - refer to clockwise and counter clockwise rotation of light,
respectively
R & S refer to structural assignments according to priority rules
R: Rectus S: Sinister
D & L refer to structural assignments according to priority rules
 Fischer projections I12

and multiple chiral centers

CHO CHO
HC OH HO CH
erythrose
R S
HC OH HO CH
R S
CH2OH CH2OH
(-) D (+) L diastereomers
enantiomers
CHO CHO
HO CH HC OH
HC OH HO CH
threose
CH2OH CH2OH
(-) D (+) L

Fischer projections: Vertical lines point in (away), horizontal lines point out
Molecule with n chiral centers: 2n Stereoisomers, 2n/2 pairs of Enantiomers
 CHO CHO I13
HC OH HO CH glyceraldehyde
CH2OH CH2OH
(+) D (-) L

1 CH3 2 CH3 2,3-

H Cl dichlorobutane
S Cl H
R
Cl H enantiomers H
S R Cl
CH3 CH3

CH3 CH3 diastereomers

H Cl Cl H
H Cl meso Cl H
compounds
CH3 CH3
3 4
 I14
Sequence of Priority Rules
• Rank 4 groups on chiral C by priority
– Highest atomic #, highest priority
– Two isotopes of same element present, one with higher
mass  higher priority
– If #1 can’t be applied, move out along C-chain until a
group of higher priority reached
– Double or triple bonds indicate groups on those bonds
are duplicated or triplicated, e.g.
O O
R-C-CH3 Equivalent to R-C-CH3
O
CC

R-C CH3 Equivalent to R-C-CH

CC
 I15
Glyceraldehyde – Fischer projections
CHO CHO

H C OH HO C H

CH2OH CH2OH
(+) D (-) L

CHO CHO
H HO
OH H

CH2OH CH2OH

2 2
4 1
1 4

3 3
(R) (S)
 I16

Limonene

CHH CCH CCH

CHH
4
3 H 2
CHH CHH CHH CHH

1
4 H

With H pointing toward rear = (S)(-) If H is pointed toward you = (R)(+)

 I17
2,3-dihydroxybutanoic acid – Fischer projections
2 COOH COOH
COOH COOH
4 R S R S
1 H OH HO H
H OH HO H
2
R 3 1 S 4 S R
H OH HO H HO H H OH
3
CH3 CH3 CH3 CH3

A
Enantiomers Enantiomers

4 2
1 2
2 4 1
1 4

3 3 3
(R) (S)
 I18
Morphine
C,H,O
HO
HO
H

C,C,H
O
H

H
N 2 1
HO H
CH3
H
3
IUPAC Name: (S)

7,8-didehydro-4,5-epoxy-17-methylmorphanin-3,6-diol
 E1

Enzyme Classification
• Introduction
– Enzymes (E) do not cause new types of organic
reactions. They do speed up reactions that are
thermodynamically and mechanistically possible
– Furthermore, E control reactions in 2o met just as they
do in 1o met
– Brief look at pathways and literature reveals E often
not identified
– Thousands E involved; can’t learn all
– Must become familiar with and be able to identify
general E reactions
– A rational scheme for E classifications established in
1961 and it helps us identify E types
 E2
Enzyme function and properties
• Enzyme are proteins (or are they always?)
• They do not allow new kinds of organic
reactions
• They are not “Magic Boxes”, rather, they
are “Black Boxes” that speed up allowed
organic reactions and allow them to occur
with great specificity
 E3
Enzyme function and properties
• Enzymes lower kinetic barriers of reactions
• Speed up reactions up to 1012 fold
• They do not change the equilibrium constant
or G of a reaction
• Assume A B with the enzyme ABase
• Energetics on a reaction coordinate:
Without Enz Activation E w/o enz
A Activation E w/ enz
E
B G same w/ or w/o enz
With Enz

Extent of Reaction
 E4
Enzyme function and properties
Without Enz Activation E w/o enz
E A Activation E w/ enz

B G same w/ or w/o enz

With Enz
Extent of Reaction
• Enz does not change G or Keq for rxn
[B] [B]
= Keq = G = - RT ln (Keq)
[A] [A]
w/o Enz w/ Enz
k1
• For A B, k1/k-1 = Keq
k-1
• Thus, if k1 + enz > k1 – enz, k-1 + enz > k-1 – enz by
the same amount
 Assumptions about enzymes E5

• Each enzyme can only catalyze a specific

reaction
• Each enzyme is selective to a specific substrate
or small group of substrates
• Each enzyme will only produce one product or a
small group of products
• Each enzyme only accepts one enantiomer and
produces a stereo specific product
• 3 dimensional structure of an enzyme and co-
factors make reaction state intermediate
possible
• In many cases acid-base catalysis is key to
enzyme function using acidic and/or basic amino
acids
Co-substrates that are very common E6
• ATP – Energy source
• CoASH – Activated group carrier
• NAD(P)H – Reducing power
• For example: Use of ATP to drive a reaction
(ligase reaction)
ROH + ATP RO-AMP + PPi
RO-AMP + HY RY + AMP
• 2 reactions on one enzyme to make
ROH + HY RY favorable
• AMP is a good leaving for the nucleophillic
attack of Y on R
 E7
All enzymes fall into six large classes
E classifications established in 1961
1. Oxidoreductases: Catalyze redox reactions. Transfer e-
from donor (oxidized) to acceptor (reduced).
2. Transferases: Transfer groups from donor to acceptor.
3. Hydrolases: Catalyze hydrolysis of C-O and C-N bonds.
4. Lyases: Non-hydrolytic removal of a group (e.g. NH2)
often forming a double bond. Or addition of a group
across a double bond.
5. Isomerases: Transform substrate into an isomer.
6. Ligases (Synthetases): Condensing E. Catalyze union
of two molecules using ATP or other nucleoside
triphosphate.
Even self splicing RNA fits. It has a hydrolase and transferase
activity
 E8
1. Oxidoreductases

• Oxidation-Reduction (Redox) reactions

Oxidation: Loss of electrons from a chemical

Reduction: Gain of electrons by a chemical

Example of how redox reactions work:

reaction on the next 2 slides catalyzed by
Alcohol Dehydrogenase
 E9

CH3CHO + NADH + H+  CH3CH2OH + NAD+

Half reactions (Written as reduction reactions)
NAD+ + 2e- + H+  NADH o = -0.32 v
CH3CHO + 2e- + 2H+  CH3CH2OH o = -0.20 v

– Electrons will flow from the compound with the

more negative o (reductant) to the compound
with the more positive o (oxidant)
– In this case, the reductant in NADH and the
oxidant is acetaladehyde. Note: the reductant is
oxidized in the reaction and the oxidant is reduced in
the reaction
 E10

Balancing the reaction (from the half reactions)

NADH  NAD+ + 2e- + H+ o = -0.32 v
CH3CHO + 2e- + 2H+  CH3CH2OH o = -0.20 v

CH3CHO + NADH + H+  CH3CH2OH + NAD+ (balanced rxn)

Free Energy: Nernst equation G = -n F o = -(2)(23)(0.12) =

- 5.5 kcal mol-1 = - 23 kJ mol-1

F = Faraday’s Constant = 23 Kcal mol-1v-1

n = number of electrons = 2
o = o (oxidant) - o (reductant) = (-0.2)-(-0.32) = 0.12V
 E11
Classes of oxidoreductases
A. Dehydrogenases. H added or removed, O2 not
involved
(Example from previous slides)

NADH + H+ NAD+
H H
O Alcohol dehydrogenase H OH
CH3 CH3
- G
Acetaldehyde Ethanol
+ G
Used to detoxify EtOH. Goes in direction of + G if EtOH
concentration is high enough
 E12
B. Oxidases. Oxygen (O2) serves as the e-
acceptor; either H2O or H2O2 formed.
Generally: very negative G. O2 very electron poor.
Cytochrome C oxidase is a good example

CoQ Oxidized ½ O2
Cytochrome C
Cytochrome C Cytochrome C
Reductase oxidase
Reduced
CoQH2 H 2O
Cytochrome C

O2 is the e- acceptor
CoQH2 is the donor via Cytochrome C (and Cyto C
Reductase)
e-’s came from NADH in the respiratory e- transport
chain
 E13

Polyphenol oxidases (fungi and higher plants)

Cause browning of cut apples, potatoes, avacados
OH O
OH
phenolase O
+ ½ O2 + H2O
catechol o-quinone
O not added to substrate ; H+, e- transferred to O2
Note: reaction inhibited by low pH because first
step is base catalyzed
Thus, lemon juice stops browning of guacamole
OH OH

OH O-
+ H+
 E14

Peroxidases: oxidases that use H2O2 (O) as e-

acceptor
O
OH
peroxidase
+ H 2 O2 + 2H2O

O
OH
p-hydroquinone p-quinone

Catalases: oxidases that use H2O2 (O) as donor

and acceptor
H 2 O2 + H 2 O2 catalase 2H2O + O2
 E15

C. Oxygenases. Oxygen incorporated

into substrate as oxidation occurs.
Monooxygenases (mixed-function
oxygenases) One O atom in product,
other in H2O. Most are cytochrome
P450s. To be covered in more detail
later.
Good example = phenylalanine Blood test for PKU
hydroxylase a deficiency of which
causes phenylketonuria (PKU)
COO-
tyrosine
Phenylalanine
C C NH3+ hydroxylase HO C
H2
NH3+
H2 H + O2 + NADPH + H+ COO-
phenylalanine + NADP+ + H2O
 E16

Dioxygenases and lipoxygenases from plants: a

good example of O2 incorporation. It catalyzes
addition of O2 to double bonds in linoleic (18:2) +
linolenic (18:3) acids, etc to form hydroperoxides.
Bond cleavage by other enzymes occurs to give
series of fragments many of which are volatile
flavour products

Overall reaction involves a peroxidation of the cis,

cis-1,4-pentadiene portion of the FA and the
formation of a cis,trans-1,3-butadiene
hydroperoxide
 E17

cis cis
13 12 H H 10 H HH H
H 9 H
R R .
11 R R
O=O H H H

H cis H
H trans
R
RCH
RCH H

hydroperoxide O
Note: one carbon shift of double O
bond changes stereochemistry. H
Can become a chain reaction if H comes from a different lipid
 E18

D. Reductases. A somewhat non-specific term.

Generally the reverse of a dehydrogenase,
where an apparent dehydrogenase reaction
is shifted to the left. If the principal
component in the reaction is reduced (and
usually NAD(P)H is oxidized), we sometimes
call the enzyme a reductase, i.e. nitrate
reductase, glutathione reductase or
cytrochrome c reductase (above).

H+ + NO3 + NADPH NO2- + NADP+ + H2O

Nitrate
Reductase
 E20
Oxidoreductase classifications

• Dehydrogenase: Loss of H

• Oxidase: H to O acceptor

• Oxygenase: O is added

• Reductase: O is removed or H is added

 2. Transferases E20

Do not involve oxidation or reduction. Transfer

complete groups containing C, N, P or S from
one substrate to another (e.g., CH3, NH3, PO4,
OCH3)
A. Kinases. Transfer of Pi from ATP to an
acceptor. Two types recognized:
(i) Transfer among high energy compounds
Creatine
ADP + creatine-P ATP + creatine
kinase, Mg++
Creatine phosphate is a high energy carrier and energy
storage source
ATP can now be used for energy in the cell. Keeps
[ATP] constant in cells
 E21
(ii) phosphate transfer with formation
of low energy compounds

O
O OH O OH
O Hexokinase
+ ATP -O P O
OH
HO OH Mg2+
HO OH
O O
H H
α-D-Glucose Glucose-6-Phosphate

• P to sugar. Good leaving group for further reaction

• P to protein. Activation of many enzymes and
signalling factors
B. Phosphorylases. Transfer glycosyl residues E22
from starch (plant + animal) to Pi

Starch
Starch + nPi n G-1-P
phosphorylase

Note: ATP is not involved, but energy in the

glycosidic bond in starch is largely
preserved. The reverse reaction can be
demonstrated in vitro, but it does not occur
readily in vivo. Why?
– What happens to G-1-P?
– What is [Pi] in vivo?
Instead starch synthesis via:
Starch
UDP-Glu + Glun Synthetase Glun+1 + UDP
 E23

C. Aminotransferases. Transamination reactions,

i.e. transfer –NH2 from one compound to another.
Specified by two amino acids involved or by the
donor.

COOH COOH
COOH CH2 COOH
CH2 glu-asp
CH2 + CH2 CH2 + CH2
O aminotransferase CH-NH2
CH-NH2 O
COOH COOH COOH
COOH
Glutamate Oxaloacetate aa-ketoglutarate Aspartate

Note: Glutamate is the most common donor of NH2 groups.

In primary metabolism this reaction goes right to left (Asp to Glu).
Then Glu feeds the NH2 to other substrates.
 E24

D. Methyl transferases. Transfer –CH3 groups,

also called methylferases. S-adenonsyl-
methionine (SAM) is the primary source of –CH3
groups in methylation reactions.

CH3
NH3+ +
H3C N CH3
CH2 3 SAM 3(s-adenosylhomocysteine) CH
2
CH2 CH2
methyl transferase
O O
R R
EtOH Amine R could be a lipid or H Choline
 E25

E. Acetyltransferases
– Transfers acetyl groups from acetyl CoA.

(CH3)3
+
(CH3)3 O N
+
N H3C S CoA HS CoA CH2
CH2 CH2
CH2 choline acetylase O
OH O
Choline
CH3
Acetylcholine
Regeneration of acetylcholine for neurotransmission reactions.
In Neurotransmission reactions, acetylcholine hydrolyzed by
acetylcholine esterase
 E26

3. Hydrolases
Catalyze hydrolysis by addition of water

A. Glycosidases. Break glycosidic bonds; the

fundamental linkage in all poly-saccharides, and in
most di- and oligo-saccarides.

Invertase splits sucrose:

(+) Sucrose + H2O (+) glucose + D (-) Fructose
invertase
 E27
ß-glucosidase splits sugar from a cyanogenic
glucoside etc.
OH OH

b-glucosidase + glucose
+ H 2O

H C O Glu H C OH
C N C N

Dhurrin The aglycone

Cyanogenic glycosides common in plants (made from Tyr). Aglycones

are a feeding deterrents and antibiotics
 E28
ß-galactosidase splits sugar from an
indole to form a blue colored
compound
GAL-O Cl HO Cl
Br Br
+ H2O + GAL
N N

X-gal Blue colored

indole
 E29

B. Phosphatases. Hydrolyze esters of

phosphoric acid. Large, complex groups with
3 broad sub-groups:
a) Alkaline phosphatases (optimum pH ~ 9)
b) Neutral phosphatases (optimum pH ~ 5-6)
c) Acid phosphatases (optimum pH ~ 3-4)
 E30

Monophosphatases (esterases) hydrolyze

monophosphoric esters e.g. in Calvin Cycle

P OCH3 CH2-O-P P OCH3 CH2OH

O O
H H
+ H20 Phosphatase + Pi
H OH H OH

OH H OH H

Fructose - 1,6 - bis Phosphate Fructose - 6- Phosphate

 E31

Diphosphatases (Phosphodiesterases) include

DNAses, Cyclic AMP phospho-diesterase, and
snake venom diesterases. Self splicing RNA has
this activity also.
H2 O
C Adenine
O O O P O CH2 Adenine
P-Diesterase O
O
O P
O H20 H+
O OH
Stops second messenger signals OH OH
Cyclic AMP
(adenosine 3,5-monophosphate) AMP
 E32

C. Lipases
– Hydrolyze triglycerides (fats) to glycerol and
free fatty acids.
O R CH2OH
R O H + 33 HO
2O
lipase CHOH
+ 3 Fatty acids
O R CH2OH

D. Amylases
– Hydrolyze starch in storage cells to maltose
units:
amylase
Starch + nH2O nMaltose

maltase
Maltose + H2O 2 Glucose
 E33

– α-amylase cleaves α 14 bonds of amylose

and amylopectin to yield maltose and limit
dextrins.
– β-amylase removes maltose units at the non-
reducing end of the glucan. Degrades
amylose completely but amylopectin to 1  6
bonds.
– Isoamylase cleaves amylopectin at 1  6.

Used in brewing: Each has a different temperature

optimum that is used by brewers to control sugar release
 E34

F. Protease
– Cleave peptide bonds.
R
R N COOH
H
H2N O

– Peptidase hydrolyze peptides at terminal

amino acids (exopeptidases). Both N and C
peptidases.
– Proteinases can cleave at internal peptide
bonds (endopeptidases), i.e. trypsin.
 E35

4. Lyases
Catalyze non-hydrolytic fission of a group from
the substrate. e.g. H2O, CO2, NH3. Condensation
also occurs.

A. Decarboxylase
COOH
Lysine
H2N (CH2)4 NH2 H2N (CH2)5 NH2 + CO2O
decarboxylase
Lysine Cadaverine
 E36
B. Aldolase Calvin cycle (gluconeogenesis)
O
1 CxH2OH 3
4 HC 6
Aldolase 5 O OP
5 HaCOHb + 2 C=O PO H 2 O Hb
a
3 CH2OP 4 1
6 CH2OP O OH
xH
Glycolysis (Fission)
C. Lyase (deaminase)
– For example deamination by PAL:

phenylalanine
C COOH C C COOH + NH3
H2 ammonia lyase H H
NH2
Cinnamic acid
Tyrosine
Phenylalanine
 E37
D. Fumarase (hydrases, hydratases)
– For example in the TCA cycle:

COO- COO-
CHOH fumarase CH OH
+ H2O2
CH2 CH
COO- COO-
Matate
Malate Fumarate
Fumarate

No fission occurred. It was a condensation, i.e., H2O was removed

without fission of a bond. Reverse would have been addition of H2O
w/o hydrolysis.
In summary: Lyases are decarboxylases, aldolases,
some synthases, some cyclases, hydrases and
hydratases via condensations or non-hydrolytic
cleavages
 E38
5. Isomerases
Very simple group – no net changes in redox or
chemical functionality
– Includes racemases, epimerases, mutases

Phosphogluco-
(A) G-6-P isomerase
F-6-P

triosphosphate
(B) DHAP isomerase GAP
 E39
6. Ligases
– Condensing (transferring) enzymes. Performs
formation of bonds between two substrates using
ATP or other high energy bond. Also called
“synthetases” (not synthases which are generally
lyases).

(A) Carboxylase with carboxylation of pyruvate as

an example. Provides OAA in TCA cycle.
COO-
CH3
CO2 pyruvate carboxylase
O + CH2
COO- O
ATP ADP
pyruvate
COO-
oxaloacetate
 E40
B. Synthetase with formation of an amide by
incorporation of NH4+ into an amino acid as an example.
Stores N in plants
COO- COO-
CHNH2 glutamine synthetase CHNH2
Glu CH2 + NH4+
CH2 Gln
(or Asp) CH2 CH2 (or Asn)
ATP ADP
COO- CONH2

Acetyl-CoA synthetase (Acyl-X synthetases)

– Catalyzes a major reaction for primary and secondary
metabolism. ATP drives formation of a high energy
thioester bond O
synthetase
H3C COO- + HS CoA H3C S CoA
Acetic acid ATP ADP
AMP Acetyl CoA
Mg++ + PPi
K+
 E41

Mechanism of Acetyl-CoA synthetase

1. CH3COO- + ATP Acetyl-AMP + PPi

2. HSCoA + Acetyl-AMP Acetyl-CoA + AMP

AMP on the acetyl is a good leaving group.

Allows Nucleophilic attack of HSCoA on Acetyl to form
the product

Other Ligases: DNA ligase, some RNA splicing

 E42

Every enzyme is given four numbers, e.g.

PAL, phenylalanine ammonia lyase is
E.C.4.3.1.5
Where:
• E.C. stands for enzyme classification
• 4 = main class, lyase
• 3 = subclass, lyases
• 1 = sub-subclass, ammonia lyases
• 5 = enzyme series within the sub-subclass, in this
case PAL, indicating phenylalanine as the
acceptor or substrate from which ammonia is
removed.
 E43

Key to Numbering and Classification of

Enzymes
1. Oxidoreductases
1.1 Acting on the CH-OH group of donors
1.1.1 With NAD or NADP as acceptor
1.1.2 With cytochrome as an acceptor
1.1.3 With oxygen as acceptor
1.1.4 With other acceptors
1.2 Acting on the aldehyde or keto-group of donors
1.2.1 With NAD or NADP as acceptor
1.2.2 With cytochrome as an acceptor
1.2.3 With oxygen as acceptor
1.2.4 With lipoate as acceptor
1.2.99 With other acceptors
 E44

1.3 Acting on the CH-CH group of donors

1.3.1 With NAD or NADP as acceptor
1.3.2 With cytochrome as an acceptor
1.3.3 With oxygen as acceptor
1.3.99 With other acceptors
1.4 Acting on the CH-NH2 group of donors
1.4.1 With NAD or NADP as acceptor
1.4.3 With oxygen as acceptor
1.5 Acting on the C-NH group of donors
1.5.1 With NAD or NADP as acceptor
1.5.3 With oxygen as acceptor
1.6 Acting on reduced NAD or NADP as donors
1.6.1 With NAD or NADP as acceptor
1.6.2 With cytochrome as an acceptor
1.6.4 With a disulfide compound as acceptor
 E45
1.6.5 With a quinone or related compound as acceptor
1.6.6 With a a nitrogenous group as acceptor
1.6.99 With other acceptors
1.7 Acting on other nitrogenous compounds as donors
1.7.3 With oxygen as acceptor
1.7.99 With other acceptors
1.8 Acting on sulphur groups of donors
1.8.1 With NAD or NADP as acceptor
1.8.3 With oxygen as acceptor
1.8.4 With a disulfide compound as acceptor
1.8.5 With a quinone or related compound as acceptor
1.8.6 With a a nitrogenous group as acceptor
1.9 Acting on haem groups of donors
1.9.3 With oxygen as acceptor
1.9.6 With a a nitrogenous group as acceptor
 E46

1.10 Acting on diphenols and related substances as donors

1.10.3 With oxygen as acceptor
1.11 Acting on hydrogen peroxide as donor
1.12 Acting on hydrogen as donor
1.13 Acting on single donors with incorporation of oxygen
(oxygenases)
1.14 Acting on paired donors with incorporation of oxygen into
one donor (hydroxylases)
1.14.1 With NAD or NADP as acceptor
1.14.2 With ascorbate as an acceptor
1.14.3 With reduced pteridine as one donor
 E47
2. Transferases
2.1 Transferring one-carbon groups
2.1.1 Methyltransferases
2.1.2 Hydroymethyl-, formyl- and related transferases
2.1.3 Carboxyl- and carbamoyltransferases
2.1.4 Amidinotransferases
2.2 Transferring aldehyde or ketonic residues
2.3 Acyltransferases
2.3.1 Acyltransferases
2.3.2 Aminoacyltransferases
2.4 Glycosyltransferases
2.3.1 Hexosyltransferases
2.3.2 Pentosyltransferases
2.5 Transferring alkyl or related groups
 E48
2.6 Transferring nitrogenous groups
2.6.1 Aminotransferases
2.6.2 Oximinotransferases
2.7 Transferring phosphorus-containing groups
2.7.1 Phosphotransferases with an alcohol group as
acceptor
2.7.2 Phosphotransferases with an carboxyl group as
acceptor
2.7.3 Phosphotransferases with a nitrogenous group as
acceptor
2.7.4 Phosphotransferases with a phospho-group as
acceptor
2.7.5 Phosphotransferases, apparently intramolecular
2.7.6 Pyrophosphotransferases
2.7.7 Nucleotidyltransferases
2.7.78 Transferases for other substituted phospho-groups
 E49
2.8 Transferring sulphur-containing groups
2.8.1 Sulphurtransferases
2.8.2 Sulphotransferases
2.8.3 CoA-transferases

3. Hydrolases
3.1 Acting on ester bonds
3.1.1 Carboxylic ester hydrolases
3.1.2 Thioester hydrolases
3.1.3 Phosphoric monoester hydrolases
3.1.4 Phosphoric diester hydrolases
3.1.5 Triphosphoric monoester hydrolases
3.1.6 Sulphuric ester hydrolases
3.2 Acting on glycosyl compounds
3.2.1 Glycoside hydrolases
3.2.2 Hydrolyzing N-glycosyl compounds
 E50

3.2.3 Hydrolyzing S-glycosyl compounds

3.3 Acting on ether bonds
3.3.1 Thioether hydrolases
3.4 Acting on peptide bonds (peptide hydrolases)
3.4.1 α-Amino-acyl peptide hydrolases
3.4.2 Peptidyl-amino-acid hydrolases
3.4.3 Dipeptide hydrolases
3.4.4 Peptidyl-peptide hydrolases
3.5 Acting on peptide bonds (peptide hydrolases)
3.5.1 In linear amides
3.5.2 In cyclic amides
3.5.3 In linear amidines
3.5.4 In cyclic amidines
3.5.5 In cyanides
3.5.99 In other compounds
 E51

3.6 Acting on acid-anhydride bonds

3.6.1 In phosphoryl-containing anhydrides
3.7 Acting on C-C bonds
3.6.1 In ketonic substances
3.8 Acting on halide bonds
3.8.1 In C-halide compounds
3.8.2 In P-halide compounds
3.9 Acting on P-N bonds

4. Lyases
4.1 Carbon-carbon lyases
4.1.1 Carboxy-lyase
4.1.2 Aldehyde-lyase
4.1.3 Ketoacid-lyase
 E52

4.2 Carbon-oxygen lyases

4.2.1 Hydro-lyase
4.2.99 Other carbon-oxygen lyases
4.3 Carbon-nitrogen lyases
4.3.1 Ammonia-lyase
4.3.2 Amidine-lyases
4.4 Carbon-sulphur lyases
4.5 Carbon-halide lyases
4.99 Other lyases

5. Isomerase
5.1 Racemases and epimerases
5.1.1 Acting on amino acids and derivatives
5.1.2 Acting on hydroxyacids and derivatives
5.1.3 Acting on carbohydrates and derivatives
5.1.99 Acting on other compounds
 E53
40
5.2 Cis-trans isomerases
5.3 Intramolecular oxidoreductases
5.3.1 Interconverting aldoses and ketoses
5.3.2 Interconverting keto- and enol-groups
5.3.3 Transposing C=C bonds
5.4 Intramolecular transferases
5.3.1 Transferring acyl groups
5.3.2 Transferring phosphoryl groups
5.3.3 Transferring other groups
5.5 Intramolecular lyases
5.99 Other isomerases

6. Ligases
6.1 Forming C-O bonds
6.1.1 Amino-acid RNA ligases
6.2 Forming C-S bonds
6.2.1 Acid-thiol ligases
 E54

6.3 Forming C-N bonds

6.3.1 Acid-ammonia ligases (amide synthetases)
6.3.2 Acid-amino-acid ligases (peptide synthetases)
6.3.3 Cyclo-ligases
6.3.4 Other C-N ligases
6.3.5 C-N ligases with glutamine as N-donor
6.4 Forming C-C bonds

References
1. T.E. Barman (1969). Enzyme Handbook. (A catalogue of enzyme data).
Springer-Verlag. I and II; (1974). Supplement I
2. Enzyme Nomenclature. (1973). Recommendations (1972) of the Commission
on Biochemical Nomenclature on the Nomenclature and Classification of
Enzymes together with their Units and the Symbols of Enzyme Kinetics. Elsevier
3. Nomenclature of Multible Forms of Enzymes. (1978). IUPAC-IUB Commission
on Biochemical Nomenclature (CBN). Recommendations 1976. Eur. J.
Biochem. 82, 1.