Department of Chemistry and Biochemistry University of Lethbridge
Biochemistry 3300
I. Introductory Concepts Enzyme Nomenclature & Experimental Appr...
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
- 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
