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Home , Enzyme Commission number

Department of Chemistry and Biochemistry 3300 University of Lethbridge

I. Introductory Concepts Nomenclature & Experimental Approaches

Biochemistry 3300 Slide 1 Enzyme Nomenclature

Early were assigned arbitrary names (typically before the specific reaction being catalyzed was known) when discovered

eg. - dismutation of H2O2 to H2O and O2 * - (Asp), # - Protease, Endopeptidase - 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 Alcohol:NAD+ - oxidation of alcohols Urea - of Urea DNA dNTP:DNA dNMPtransferase - polymerization of nucleotides Donor:Acceptor methyltransferase - 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 Recommended name ? Enzyme Commission number: (2) 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 13C-glycogen signal increases as 13C-glucose signal decreases

Can now follow metabolic conversion within intact cells (favourable cases) T30

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