Enzymes lecture 12 Flashcards
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PHSYCHROPHILES (COLD)
Life > 70% water
• 70% Oceans are <5 oC (90% by volume)
• 75% Earth cold ecosystems
• Temperature affects: Species distribution, Abundance & Survival
• Organisms shut down growth to switch on adaptive mechanisms
general response to the cold
- shock/ stress proteins
- enzyme modification
- antioxidants
- degradation of mitochondria
- change membrane composition
Physiology of species that live in the cold
- high metabolic rates vs temperature species
- high molecular weight proteins inhibit ice formation
- modification of metabolism, eliminate or mask nucleators
- accumilation cryoprotectants FR
- manufacture the above to initiate freezing
Freeze tolerance
- ice nucleating agents (INA’S)
- ployols
- sugars
- antifreeze proteins - to lower the freeezing point of water
- recrystallization inhibitors prevent ice recrystalizing in frozen tissues
Freeze resistance strategy 3pts
- Mask internal nucleators
- reduce water content
- avoid external nucleates
How do enzymes evolve in the cold?
- Reduction in the activation energy
- High catalytic efficiency
- Weak thermal stability
- Catalytic structure identical
- Reduction in Proline and Arginine residues
- Increase or clustering of glycine residues
- Weakening of intramolecular forces
- Increased solvent interactions
- Decrease numbers of charged residue interactions and disulphide bonds
Cold shock response
- Specific pattern of gene expression in response to abrupt changes to lower temperatures
- Specific set of cold shock protein, repression of heat shock proteins
- Continued synthesis of proteins involved in transcription and translation
- Maintain the fluidity of the membrane (inducible desaturases)
General microbial responses
- maintain structural integrity, protein, membranes, ribosomes
- cell membrane composition
- specific pattern of gene regulation
- elevated levels of enzymes
Specific microbial responses
- Cold shock response; production of up to 50 proteins
- CSPs stabilize mRNA & reactivate protein production
- Membrane permeases less sensitive to low temp inactivation
- Osmotic stress – accumulate solutes
Psychrophiles
- An organism that reproduces and grows optimally at low temperature -10 to 20°C / -15 to 10°C
- Key Arctic and Antarctic oceans, Ice sheets at depths of kilometers
- Arthrobacter sp., Psychrobacter sp.
- Psychrophiles are adapted to function at low temperatures and the enzymes they possess can be denatured at moderate temperatures
- They also exhibit a wide range of other adaptations
- Psychrotolerant organisms are mesophiles that can survive at low temperatures but grow suboptimally
Enzymes from psychrophilic organisms
high catalytic efficiency at low and moderate temperatures but are rather thermolabile.
Due to their high specific activity and their rapid inactivation at temperatures as low as 30°C, they offer, along with the producing micro-organisms, a great potential in biotechnology.
The molecular basis of the adaptation of cold α-amylase, subtilisin, triose phosphate isomerase from Antarctic bacteria and of trypsin from fish living in North Atlantic and in Antarctic sea waters have been studied. The comparison of the 3D structures obtained either by protein modelling or by X- ray crystallography (North Atlantic trypsin) with those of their mesophilic counterparts indicates that the molecular changes tend to increase the flexibility of the structure by a weakening of the intramolecular interactions and by an increase of the interactions with the solvent. For each enzyme, the most appropriate strategy enabling it to accommodate the substrate at a low energy cost is selected. There is a price to pay in terms of thermosensibility because the selective pressure is essentially oriented towards the harmonization of the specific activity with ambient thermal conditions. However, as demonstrated by site-directed mutagenesis experiments carried out on the Antarctic subtilisin, the possibility remains to stabilize the structure of these enzymes without affecting their high catalytic efficiency.
Psychrophilic enzymes: a thermodynamic challenge
Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0–30°C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold α-amylase, β-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost.
Molecular adaptations of enzymes from psychrophilic organisms
The dominating adaptative character of enzymes from cold-evolving organisms is their high turnover number (kcat) and catalytic efficiency (kcat/Km), which compensate for the reduction of chemical reaction rates inherent to low temperatures. This optimization of the catalytic parameters can originate from the highly flexible structure of these proteins providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Molecular modelling of the 3-D structure of cold-adapted enzymes reveals that only subtle modifications of their conformation can be related to the structural flexibility. The observed structural features include: 1) the reduction of the number of weak interactions involved in the folded state stability like salt bridges, weakly polar interactions between aromatic side chains, hydrogen bonding, arginine content and charge-dipole interactions in α-helices; 2) a lower hydrophobicity of the hydrophobic clusters forming the core of the protein; 3) deletion or substitution of proline residues in loops or turns connecting secondary structures; 4) improved solvent interactions with a hydrophilic surface via additional charged side chains; 5) the occurence of glycine clusters close to functional domains; and 6) a looser coordination of Ca2+ ions. No general rule emerges from the molecular changes observed; rather, each enzyme adopts its own strategy by using one or a combination of these altered interactions. Enzymes from thermophiles reinforce the same type of interactions indicating that there is a continuity in the strategy of protein adaptation to temperature
Pseudoalteromonas haloplanktis
Pseudoalteromonas haloplanktis TAC125 is a fast growing gammaproteobacterium isolated from an Antarctic coastal sea water sample collected near the French Antarctic station Dumont d’Urville, Terre Adelie, Antarctica.
• No structural alteration in enzyme catalytic centre
• Several in overall structure (explaining weak thermal stability)
• Loss of stability provides required active site mobility
Cold Adapted Archaea
Methanogenium frigidum & Methanococcoides burtonii
• Largest and greatest diversity of archaea in cold environments
• Methanogens – produce CH4
• In comparison with warm methanogens
– 5 unique genes (nucleic acid binding proteins, RNA helicase)
• M. burtonii 560 proteins expressed at 4 oC; 44 differentially expressed at 23 oC
• M. frigidum has CSPs (not found in heat loving archaea)
