Department of Chemistry and Biochemistry Biochemistry 3300

University of Lethbridge

I. Introductory Concepts
Enzyme Nomenclature &
Experimental Approaches

Biochemistry 3300 Slide 1

 Enzyme Nomenclature

Early enzymes were assigned arbitrary names (typically before the

specific reaction being catalyzed was known) when discovered
eg. Catalase - dismutation of H2O2 to H2O and O2
Pepsin* - Protease (Asp), Endopeptidase
Trypsin# - Protease, Endopeptidase
Lysozyme - lyses bacterial cell walls

Rapid growth in rate of discovery of enzymes led

to development of nomenclature rules (1992)!

International Union of Biochemistry and

Molecular Biology (IUBMB) propose
Enyzme Commision Nomenclature

* Greek pepsis = digestion

#
Greek tryein = to wear down

Biochemistry 3300 Slide 2

 Enzyme Nomenclature

Enzymes are classified and named according to

the nature of the chemical reactions they catalyze.

Enzymes are assigned two names and a classification number.

Recommended name: everyday use (often previous trivial name)

Systematic name:name of substrate(s) + name of reaction catalyzed

(group classification) with –ase suffix

Enzyme commission (E.C.) number: 4 numbers that uniquely categorize

each enzymatic reaction

Biochemistry 3300 Slide 3

 Enzyme Nomenclature

Recommended name:
Original names OR named by appending –ase to either the:
- name of a substrate
- type of catalytic reaction

Systematic name:
Substrates are listed first (colon separated) followed by the
type of catalytic reaction with the suffix -ase

Some examples:

Recommended Systematic Reaction

Alcohol dehydrogenase Alcohol:NAD oxidoreductase
+
Urease Urea amidohydrolase
DNA polymerase dNTP:DNA dNMPtransferase
Methyltransferase Donor:Acceptor methyltransferase
- oxidation of alcohols
- hydrolysis of Urea
- polymerization of nucleotides
- methyl group transfer

Biochemistry 3300 Slide 4

 Enzyme Classification
(EC Numbers)
Group Number: six 'groups' of catalyzed reaction types:

Remaining three numbers describe all possible subclasses

http://www.chem.qmul.ac.uk/iubmb/enzyme/
Tipton,K.F., The naming of parts, Trends Biochem. Sci. 18, 111-115 (1993)

Biochemistry 3300 Slide 5

 Enzyme Classification

Example:
ATP + D-glucose → ADP + D-glucose 6-phosphate

Systematic name
ATP:glucose phosphotransferase
Recommended name
?
Enzyme Commission number: (2) transferase reactions
(7) phosphoryl group transfer
2.7.1.1
(1) hydroxyl group as acceptor
(1) arbitrarily assigned serial number

Biochemistry 3300 Slide 6

 Enzyme Databases
More uses for EC Numbers

EXPASY (Expert Protein Analysis System)

- http://www.expasy.ch/

The Comprehensive Enzyme information system

- http://www.brenda-enzymes.org/index.php4

KEGG: Kyoto Encyclopedia of Genes and Genomes

- http://www.genome.jp/kegg/

Biochemistry 3300 Slide 7

 KEGG Pathways
Photosynthesis – Reference pathway

Biochemistry 3300 Slide 8

 Study of Metabolic Pathways
Historically, the study of metabolism / biochemistry
can trace its roots to the study of :

(1) Wine fermentation (Pasteur & Buchners)

- conversion of sugars to alcohol (and CO2)
requires yeast factor(s)

(2) Digestive system (Beaumont & St Martin*)

- conversion of various foodstuff to simpler compounds

Drawing of Alexis St Martin's stomach

Biochemistry 3300 Slide 9

 Study of Metabolic Pathways
Three major properties are studied:
a) Sequence of reactions b) Reaction mechanisms c) Control of reactions

How do you study a metabolic pathway (in very simple terms)?

(Many) Problems to consider:

1) Which compounds in the cell are metabolites in the pathway?

How do we show a metabolite is part of a particular pathway?

2) How do you detect metabolites in the cell?

Metabolites are more diverse than proteins/nucleic acids and often present
in low concentration.

3) Have all reactions been identified?

How do we show a pathway is complete?

Biochemistry 3300 Slide 10

 Study of Metabolic Pathways

How do you study a metabolic pathway (in very simple terms)?

A) Growth studies in presence of defined nutrients

- Metabolic inhibitors or Genetic mutations perturb pathway and help in both
metabolite identification and establishing sequence of reaction

B) In vitro studies in presence of defined nutrients

- Many eucaryotic pathways are specific to certain organelles. Isolation of organelles
greatly simplifies metabolic studies.

C) Substrate labeling
- Allows direct visualization of metabolites of a pathway. Time-course studies can also
reveal sequence of reactions

In all cases, the biggest difficulty is typically the detection and identification of metabolite

Biochemistry 3300 Slide 11

 Metabolic Inhibitors
Glycolysis: first metabolic pathway characterized

How: Cell free extract (e.g. lysed yeast)

Expt 1: Normal Conditions:
Glucose → pyruvate

Expt 2: Presence of metabolic inhibitor (iodoacetate)

Glucose + iodoacetate → fructose-1,6-bisphosphate accumulates

Expt 3: Presence of metabolic inhibitor (fluoride)

Glucose + fluoride → 2- and 3-phosphoglycerate accumulate

“Chemical intuition combined with inhibition

data led to the prediction (and detection)
of the Pathways intervening steps.”

eg. chemical intuition

Fructose (ketose, 6C sugar) is produced from
glucose (aldose, 6C sugar)
→ isomerization reaction likely occurs
http://www.genome.jp/kegg/pathway/map/map00010.html

Biochemistry 3300 Slide 12

 Metabolic Inhibition (Genetics)
Metabolic Blocks can be generated by genetic manipulations.
The basic metabolic pathways in most organisms are identical

George Beadle and Edward Tatum generated (X-ray)

Arg-requiring auxotrophic mutants of N. crassa to elucidate
Arg biosynthesis pathway.

Neurospora crassa

Biochemistry 3300 Slide 13

 Pitfalls of Inhibition Studies

eg. Phenylalanine / Tyrosine metabolism

Known Mutants:

Phenylketonuria:
→ phenylpyruvate↑ (urine)
Alcaptonuria:
→ homogentistic acid (urine)

Phenylpyruvate is formed by a
secondary pathway!

Always a potential problem ...

Biochemistry 3300 Slide 14

 Metabolic Inhibition
Other (modern) methods of inhibition: - knock outs (mice or yeast)
- RNAi (silencing)

All these methods face a similar problem:

How do you detect the metabolites / intermediates?

Isolating metabolites difficult and pretty invasive

(except urine/blood samples)

Franz Knoop (1904) introduced the use of Isotopes

as tracers to study fatty acid metabolism.

Biochemistry 3300 Slide 15

 Isotopes and Metabolism
Isotopes (differing number of neutrons)
Isotopes in metabolic studies:
- NMR studies of metabolites in intact cells/organs (recent development)
- (radio)isotopes help to identify metabolites

Isotopes commonly used in NMR analysis:

Label atom(s) of substrate

(13C or 15N, usually):

Follow labeled atom over time

Biochemistry 3300 Slide 16

 Isotopes in Biochemistry (NMR)

T0
Example:

Conversion of [1-13C]glucose to
glycogen as observed by localized
in vivo 13C NMR.
T5 13
C-glycogen signal increases as
13
C-glucose signal decreases

Can now follow metabolic conversion

T30 within intact cells (favourable cases)

Biochemistry 3300 Slide 17

 Isotopes in Biochemistry
Metabolic origin of the N in heme.

Grow organism using

labeled compounds

eg. Labeled heme only

produced when grown
using labeled Gly

N atoms of heme
originate from Gly

Biochemistry 3300 Slide 18

 Isotopes in Biochemistry
Radioactive Isotopes commonly used:

Why radioisotopes?

easy to detect
amazing sensitivity

Biochemistry 3300 Slide 19

 Isotopes in Biochemistry

Radioisotope tracers: establishing

the order of metabolic intermediates
(precursor-product relations)

Pizza* → A* → B* → later products*

Chase experiment

Biochemistry 3300 Slide 20

 Modern Approaches

Systems Biology – transcriptomics, proteomics ..

Discovery based approach to identify the set of transcripts or protein in a

cell under a particular condition.

Comparisons of the transcriptome (or proteome) in the presence and

absence of a substrate can be used to identify the enzymes within a
pathway, the likely products and some indication of the sequence of
reactions

Biochemistry 3300 Slide 21

 Is this still 'hot'?

YES !!!!

Berg et al., SCIENCE (2007)

Biochemistry 3300 Slide 22