Download Enzymes - University of Lethbridge

Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 3300
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
Pepsin*
Trypsin#
Lysozyme
- dismutation of H2O2 to H2O and O2
- Protease (Asp), Endopeptidase
- 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 dehydrogenase
Urease
DNA polymerase
Methyltransferase
Alcohol:NAD oxidoreductase
Urea amidohydrolase
dNTP:DNA dNMPtransferase
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:
2.7.1.1
Biochemistry 3300
(2) transferase reactions
(7) phosphoryl group transfer
(1) hydroxyl group as acceptor
(1) arbitrarily assigned serial number
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
T30
Biochemistry 3300
C-glycogen signal increases as
13
C-glucose signal decreases
13
Can now follow metabolic conversion
within intact cells (favourable cases)
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