Biochemistry 3300 Slide 1

I. Introductory ConceptsEnzyme Nomenclature &

Experimental Approaches

Department of Chemistry and BiochemistryUniversity of Lethbridge

Biochemistry 3300

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Early enzymes were assigned arbitrary names (typically before thespecific 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

* Greek pepsis = digestion# Greek tryein = to wear down

Rapid growth in rate of discovery of enzymes ledto development of nomenclature rules (1992)!

International Union of Biochemistry and Molecular Biology (IUBMB) propose Enyzme Commision Nomenclature

Enzyme Nomenclature

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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

Enzymes are classified and named according tothe nature of the chemical reactions they catalyze.

Enzyme Nomenclature

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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 thetype of catalytic reaction with the suffix -ase

Some examples:

Recommended Systematic ReactionAlcohol dehydrogenase Alcohol:NAD+ oxidoreductase - oxidation of alcoholsUrease Urea amidohydrolase - hydrolysis of UreaDNA polymerase dNTP:DNA dNMPtransferase - polymerization of nucleotidesMethyltransferase Donor:Acceptor methyltransferase - methyl group transfer

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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)

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Enzyme Classification

Example:

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

Systematic name

ATP:glucose phosphotransferase

Recommended name

?Enzyme Commission number:

2.7.1.1

(2) transferase reactions(7) phosphoryl group transfer(1) hydroxyl group as acceptor(1) arbitrarily assigned serial number

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Enzyme Databases

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/

More uses for EC Numbers

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KEGG PathwaysPhotosynthesis – Reference pathway

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Study of Metabolic Pathways

Historically, the study of metabolism / biochemistrycan trace its roots to the study of :

(1) Wine fermentation (Pasteur & Buchners)- conversion of sugars to alcohol (and CO

2)

requires yeast factor(s)

(2) Digestive system (Beaumont & St Martin*)- conversion of various foodstuff to simpler compounds

Drawing of Alexis St Martin's stomach

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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 presentin low concentration.

3) Have all reactions been identified? How do we show a pathway is complete?

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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 organellesgreatly simplifies metabolic studies.

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

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

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Metabolic InhibitorsGlycolysis: 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 intuitionFructose (ketose, 6C sugar) is produced fromglucose (aldose, 6C sugar) → isomerization reaction likely occurs

http://www.genome.jp/kegg/pathway/map/map00010.html

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Metabolic Inhibition (Genetics)

Metabolic Blocks can be generated by genetic manipulations.

The basic metabolic pathways in most organisms are identical

Neurospora crassa

George Beadle and Edward Tatum generated (X-ray)Arg-requiring auxotrophic mutants of N. crassa to elucidate Arg biosynthesis pathway.

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Pitfalls of Inhibition Studies

Known Mutants: Phenylketonuria:

→ phenylpyruvate↑ (urine)Alcaptonuria:

→ homogentistic acid (urine)

eg. Phenylalanine / Tyrosine metabolism

Phenylpyruvate is formed by a secondary pathway!

Always a potential problem ...

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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 Isotopesas tracers to study fatty acid metabolism.

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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

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Isotopes in Biochemistry (NMR)

Example:

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

Can now follow metabolic conversionwithin intact cells (favourable cases)

T0

T5

T30

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Isotopes in Biochemistry

Metabolic origin of the N in heme.

Grow organism usinglabeled compounds

eg. Labeled heme only produced when grownusing labeled Gly

N atoms of hemeoriginate from Gly

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Isotopes in Biochemistry

Radioactive Isotopes commonly used:

Why radioisotopes?

easy to detect amazing sensitivity

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Isotopes in Biochemistry

Radioisotope tracers: establishingthe order of metabolic intermediates(precursor-product relations)

Pizza* → A* → B* → later products*

Chase experiment

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Modern Approaches

Systems Biology – transcriptomics, proteomics ..

Discovery based approach to identify the set of transcripts or protein in acell under a particular condition.

Comparisons of the transcriptome (or proteome) in the presence andabsence of a substrate can be used to identify the enzymes within a pathway, the likely products and some indication of the sequence of reactions

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Is this still 'hot'?

YES !!!!

Berg et al., SCIENCE (2007)