Module 7

Protein Function

Related to structure and folding

Rare Codon

Low level of tRNA recognize codon

Codon Optimization

Optimal codon usage for protein-production organism

tRNA

Long single-stranded RNA

3D structure from intermolecular interactions

Contain anticodon complement to specific codon

Aminoacyl-tRNA

tRNA covalently bonded to amino acid matching codon

Aminoacyl-tRNA Synthetase

Enzyme reading anticodon

Attach amino acid with ester linkage

Bacterial Ribosome

70S

Large

2 subunits: Large 50S, small 30S

Ribosome Function

Read mRNA

Recruit tRNA

Strengthen binding between codon and anticodon

Form peptide bond between amino acids

Translation Initiation

1. Initiator tRNA with fMET enter P site
2. Initiation factors assemble mRNA, tRNA, ribosomal subunit complex

Translation Elongation

1. Ribosome read mRNA 5’-3
2. Elongation factors deliver aminoacyl-tRNA to A site
3. Amino acids add to polypeptide
4. Ribosome move down codon

Translation Termination

1. Release factors enter A site
2. Ribosome break bond between polypeptide and tRNA
3. Ribosome and protein dissociate from mRNA

Protein Folding

During translation

Chaperone proteins help

Improper Protein Folding

Protein aggregation

Form insoluble inclusion bodies in cytoplasm

Kasugamycin

Bind 30S subunit (P and E sites)

Block interaction with mRNA

No initiation complex formation

Tetracycline

Bind 30S and 50S subunits (A site)

No new amino acid addition

Peptide stuck on P site tRNA

Chloramphenicol

Bind 50S subunit

Block peptide bond formation

Neomycin

Block translocation

Prevent mRNA move through ribosome (tRNA movement)

Puromycin

Resemble nucleoside

Incorporate into peptide (A site)

No new amino acid addition

Terminate translation

Enzymes

Proteins accelerating chemical reactions

Stabilize transition state

Decrease activate energy

Transition State

High energy intermediate

Between substrate and product

Unstable

Activation Energy

Energy difference between substrate and transition state

Active SIte

Specific substrate binding

Stabilize transition state

Form unstable enzyme-substrate complex

Product Inhibition

Product bind and block enzyme active site

Enzyme Kinetics

Constant enzyme concentration

Substrate concentration decrease

Product concentration increase

Enzyme Kinetics: UV/Vis Spectrophotometry

Substrate and product absorb different light wavelengths

Substrate absorbance decrease

Product absorbance increase

Ideal Wavelength: Strong product absorbance, weak substrate absorbance

Enzyme Kinetics:  β-Lactamase

β-lactamase hydrolyze β-lactam ring

Intact ring absorb at 233 nm

Hydrolyzed ring weak absorption

Absorbance decrease rate = antibiotic degradation rate

Enzyme Kinetics: Colourimetric Nitrocefin Assay

Intact β-lactam ring: Yellow

Hydrolyzed β-lactam ring: Red

Measure absorbance at 486 nm

Inhibitors

Molecules blocking enzyme activity

Reversible Inhibition

Reversible binding to enzyme

Reversible: Competitive Inhibitor

Bind active site

Block substrate binding

Reversible: Uncompetitive Inhibitor

Bind enzyme-substrate complex at allosteric site

Change active site shape

Reversible: Noncompetitive Inhibitor

Bind enzyme or enzyme-substrate complex at allosteric site

Irreversible Inhibition

Covalent modifications on enzyme

Change active site shape

Modify amino acid side chains

Inhibit enzymatic catalysis

Inhibitor Potency

Inhibitor interaction strength with enzyme

Compare to no inhibitor enzyme activity

Percent Inhibition

100% - \[vo with inhibitor / vo without inhibitor\] x 100%

Potent inhibitor: high %

No info on enzyme/inhibitor concentration

Half-Maximal Inhibitory Concentration (IC50)

Inhibitor concentration inhibiting 50% enzyme activity

% inhibition = 50%

Good inhibitor: low IC50

Enzyme Functions

Synthesize cell wall

DNA replication and transcription

Protein synthesis

Metabolic pathway regulation

Trimethoprim/Sulfamethoxazole (TMP/SMX)

Inhibit tetrahydrofolate synthesis enzyme

Inhibit bacteria growth

TMP/SMX: Dihydropteroate Synthetase

Form covalent bond between DHPP and pABA

Reversible competitive inhibition by sulfamethoxazole

Similar to pABA structure

TMP/SMX: Dihydrofolate Reductase

Convert dihydrofolate to tetrahydrofolate

Reversible competitive inhibition by trimethoprim

Similar to dihydrofolate structure

β-Lactam Antibiotics

Antibiotic group

Covalent PBP inhibition

Form complex with active serine

Weaken cell wall cause cell death

PBP

Penicillin-binding proteins

For cell wall peptidoglycan synthesis

Transglycosylase: Link sugars

Transpeptidase: Form peptide cross-links (reactive serine)

Serine β-Lactamase (SBL)

Enzyme degrading β-lactam antibiotics

Similar structure to PBP

Serine react with and hydrolyze antibiotic

SBL Inhibitors

Protect antibiotic

Contain β-lactam ring reacting with serine and inhibit SBL

No antibiotic hydrolyzation

Mutations

Change protein structure and function

DNA replication mistakes

Transfer through vertical gene transfer