i. introductory concepts - university of...
TRANSCRIPT
Biochemistry 3300 Slide 1
I. Introductory ConceptsEnzyme Nomenclature &
Experimental Approaches
Department of Chemistry and BiochemistryUniversity of Lethbridge
Biochemistry 3300
Biochemistry 3300 Slide 2
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
Biochemistry 3300 Slide 3
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
Biochemistry 3300 Slide 4
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
Biochemistry 3300 Slide 5
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 6
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
Biochemistry 3300 Slide 7
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
Biochemistry 3300 Slide 9
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
Biochemistry 3300 Slide 10
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?
Biochemistry 3300 Slide 11
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
Biochemistry 3300 Slide 12
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
Biochemistry 3300 Slide 13
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.
Biochemistry 3300 Slide 14
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 ...
Biochemistry 3300 Slide 15
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.
Biochemistry 3300 Slide 16
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 17
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
Biochemistry 3300 Slide 18
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
Biochemistry 3300 Slide 19
Isotopes in Biochemistry
Radioactive Isotopes commonly used:
Why radioisotopes?
easy to detect amazing sensitivity
Biochemistry 3300 Slide 20
Isotopes in Biochemistry
Radioisotope tracers: establishingthe order of metabolic intermediates(precursor-product relations)
Pizza* → A* → B* → later products*
Chase experiment
Biochemistry 3300 Slide 21
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