Structural Biochemistry/Cold-Adapted enzymes

Organisms that live in perpetually cold environments have enzymes that function very effectively in the cold. Some examples of this include fish, which have also evolved to develop antifreeze proteins as a way of adapting, and some prokaryotes that have to live in cold places. Recent discoveries have uncovered that further investigation of these enzymes could actually have biotechnological applications. The most commonly referred to cold-adapted enzyme is the alpha amylase from Pseudoalteromonas haloplanktis (AHA), a type of prokaryote. The ability of organisms to thrive in cold environments comes from their capacity to synthesize cold-adapted enzymes. Organisms that thrive in cold environments are called psychrophiles. These enzymes have developed a range of structural features that allows for high flexibility particularly around the active site, low-activation enthalpy, low-substrate affinity, and high specific activity at low temperatures. The study of the structure, function, and stability of cold-adapted enzymes is rudimentary in the research into protein folding and catalysis, a still developing field.

Thermal adaptation

Different organisms have evolved in different ways, causing them to adapt to different thermal environments that suits them. Thermal adaptation in extremes is particularly hard to adapt to and has a limited range that an individual organism can tolerate. Psychrophilic microorganisms, those that have adapted to the cold, are said to be able to metabolize in snow and ice at −20°C. Some psychrophilic can even proliferate at ≤0°C but are restricted to temperatures <30°C (2–5).

Since there are so many microorganisms found in the world's oceans, cold alpine regions, caves, upper atmosphere, and polar regions, a large proportion of biomass, or living organisms in a certain region, on Earth is generated at these cold temperatures in their specific areas. Organisms from the three domains of life: Bacteria, Archaea, and Eucarya, are also from these cold environments. Most cold-adapted enzymes are found to have come from prokaryotes. In order to survive, all these organisms have to be at thermal equilibrium with their surrounding environment. This comes from all the components of their cells being appropriately adapted to the cold. In order to adapt to the cold, potential mechanistic diversity and cell-specific adaptation strategies have been adopted. A general pattern that has emerged from research of these organisms is that organisms that live in permanently cold environments usually develop enzymes that help them function capably in the cold

An example of a cold-adapted enzyme which will be further studied and explained is the α-amylase from Pseudoalteromonas haloplanktis (AHA). This enzyme is the most extensively studied cold-adapted enzyme

Activity of Cold-Adapted Enzymes

Arrhenius equation below describes the rate of all reactions including enzymatic reactions.

k_cat = AKe^-E_a/^RT

k_cat = enzyme reaction rate (increases with an increase in absolute temperature (T), decreases in activation energy (Ea)

A = preexponential factor

K = dynamic transmission coefficient (generally assumed to be 1)

R = universal gas constant (8.314 J mol−1 K−1)

According to this equation, due to low temperatures from 0°–4°C, an inadequate amount of kinetic energy is available for the system to overcome barriers. Some strategies that help compensate the slow metabolic rates include: an energetically expensive strategy of increasing enzyme concentration, seasonal expression of isoenzymes in fish and nematodes, and the evolution of enzymes in which reaction rates tend to become more temperature independent and instead approach diffusion control. Cold-adapted enzymes tend to shift their optimum temperature of activity to a lower temperature with a concurrent decrease in stability. These enzymes show a high-reaction rate when they decrease their activation free-energy barrier between the ground state and transition states according to the equation below:

ΔG#=ΔH# -TΔS#

ΔG# = activation free-energy barrier

ΔH# = is the change in activation enthalpy

ΔS# = is the change in activation entropy

T = is the absolute temperature

Stability of Cold-Adapted Enzymes

The structure of cold-adapted enzymes are highly flexible which allows them to unfold at low to moderate temperatures. Researches have tried many methods to study the unfolding and folding transitions in order to determine the way they unfold and measure the kinetic and conformational stabilities of enzymes. Such methods include spectrophotometric, calorimetric, and electrophoretic methods. Experiments have been conducted using these techniques on multidomain proteins like chitobiase. Proteins have a tendency to unfold and such processes are irreversible. When large multidomain proteins unfold due to heat, these proteins tend to be kinetically driven to finish the unfolding process.

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  Structure of Chitobiase

The only example of a cold-adapted enzyme that has reversible unfolding is AHA. At temperatures of 20°C or above, AHA has reversible unfolding that is shown by 100% recovery of ΔH cal., the total amount of heat absorbed during unfolding, during a second scan after the initial thermal denaturation. Small-molecular weight enzymes unfold with the process of cooperative unfolding. Cooperative unfolding happens because of its tightly packed bulk which causes a small number of interactions between other structural elements, helping it conserve its natural state. If the limited number of interactions in cooperative unfolding were to be disturbed, a two-state unfolding may occur due to increased interactions.

In the enzyme pancreatic porcine α-amylase (PPA) and more stable mutants of AHA, the two-state unfolding process does not occur. Instead, other types of folding could come from increased ionic interactions, causing the frequency of intramolecular discrepancies to occur during folding. AHA mutants show this concept of discrepancies or divergence, causing the rate of thermal inactivation to be directly comparable to the extent of reversibility.

In the enzyme TUG-GE, at the temperatures 3°C and 12°C, AHA unfolds reversibly and shows two transitions. The transition that unfolds at a lower urea concentration belongs to the active-site region. The active site is formed by cooperative unfolding of structures, showing independent unfolding of other more stable regions, or the domains, of the protein.. The substrate-binding region of the cold-adapted enzyme is found to be the most flexible region when the unfolding starts due to its high Km. The research on AHA shows that instability of active-site region is key to heat-labile enzymes, and is an important concept in examining a broader range of cold-adapted enzymes

Kinetic Stability

The process of enzyme inactivation or denaturation is shown with kinetic stability. Most cold-adapted enzymes have a half-life of less than 20 minutes at the temperature of 50°C, some enzymes even denature at lower temperatures. In order to predict kinetic stability, it is essential to consider the magnitude of the free-energy change between the folded, or active, state and the transition state shown below by:

F K¦↔ 〖TS〗^# k¦→ D

F = folded enzyme

K = equilibrium constant

K= first-order rate constant

D = denatured state

Cold-adapted enzymes increase the rate of thermal unfolding with a decreased ΔG# shown by the equation:

ΔG#= =RT ln K

Reduced thermostability of cold-adapted enzymes could be due to low ΔH# of the folded form. A certain number of monovalent interactions need to be broken to reach transition state (TS#) and a reduction in this number causes the low number of the folded form. An example of an enzyme that has low thermostability, a direct result from increased disorder of the transition state, is the enzyme glutamate dehydrogenase. During unfolding, a key note to the decreased entropy of thermostable enzymes could have come about from the hydration of nonpolar groups. This is caused by water forming ordered structures around hydrophobic side chains thus decreasing the entropy o the system. It is vital to determine activation parameters of denaturation in order to discover a larger range of cold-adapted enzymes and their thermostable homologs.

Flexibility and Structural adaptation

X-ray structures of cold-adapted enzymes have been discovered to show the structural basis of cold adaptation by comparing these x-ray structures with homology models of proteins from mesophiles and thermophiles. An important factor to the activity and stability of a enzyme is its physiological environment. Studies have also shown that mainly marine organisms have been used to study cold-adapted enzymes. The X-ray structure of enzyme citrate synthase has been determined and compared to other enzymes of Bacteria, Eucarya, and Achaea. Due to the absence of crystal structure information, studies generally choose closely related organisms. By maximizing thermal differences and minimizing phylogenetic differences in comparative data sets, the lack of x-ray structures for enzymes can be countered.

Hydrophobic Interactions

Hydrophobic interactions between hydrophobic residues and solvent water molecules play a key role in contributing to the structural flexibility and thermostability of cold-adapted proteins.

Core hydrophobicity

Cold-adapted enzymes have amino acids that tend to be smaller and less hydrophobic than in homologs from mesophiles and thermophiles. Since Van der Waals interactions are weak, have short range, and distance sensitive, the distance between hydrophobic groups inside a protein will determine the enthalpic contributions to stabilization. Reduced van der Waals interactions and increased movement of internal groups will therefore destabilize cold-adapted enzymes. An example of a group inside a protein is Ille. Ille can pack efficiently inside the core and stabilize a protein due to tis branching and size. In the cold-adapted trypsins, citrate synthase, and AHA, fewer Ille residues can pack inside the core.

Hydrophobic interactions are strongest at room temperature due to the solubilities of hydrophobic side chains in water having it be at the minimal temperature 20°C. Through a study with 31 proteins that unfold reversibly, it was found that about 75 % had maximum stability around room temperature. This shows that hydrophobic interactions in the core of a protein play a key role in enhancing protein stability at low to moderate temperatures.

Surface hydrophobicity

A higher proportion of hydrophobic, or nonpolar, residues occur on the surfaces of cold-adapted enzymes shown by studies with x-ray structures of many enzymes. Using large-scale modeling and structural studies, similar conclusions were found that demonstrated that the mean fraction of the solvent-accessible surface, or buried surface, of the enzyme has a higher hydrophobicity in cold-adapted proteins. In the X-ray structure of the example with Ille, it revealed that Ille clusters at the subunit interface are absent in the cold-adapted enzyme while tightly packed hydrophobic clusters are present in the homolog from a hyperthermophile.

Studies have shown that hydrophobic surface residues will destabilize a protein structure due to the decreased entropy of water molecules because these water molecules form cage-like structures around nonpolar residues. It was found that at lower temperatures, the entropy gain is actually reduced due to the decreased mobility of released water molecules showing that cold-adapted enzymes may gain flexibility from and have a greater capacity to tolerate increased surface hydrophobicity.

Surface Hydrophilicity

Certain cold-adapted enzymes like trypsins and β-lactamase (55) have had an increase in surface charge, mainly negative charge. For some enzymes, the negative charge is rather high but with some positive charge located near the active site. Due to high viscosity and high surface tension of water, at low temperatures, the energetic cost of disrupting H-bond networks is high. This energetic cost may be counter by surface-charged or polar amino acids that interact with water molecules with a high dielectric constant. This would then enable proper solvation and help maintain the flexibility of the enzymes. With better solvent interaction and positive charge, flexibility may be improve at low temperatures.

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  Structure of Trypsin

Localization of acidic residues in surface patches have the possibility of producing charge-charge repulsions that cause the overall destabilization of protein structure. Sometimes the case arises where charge repulsion of acidic residues may create a high level of flexibility in the linker region, This is a major structural feature of cold adaptation in certain enzymes. Other studies have shown that cold adaptation involves a decrease in the mean fraction of solvent-accessible and buried surface that is charged. This involves other proteins from not only the Achaea family, but also Bacteria family causing and increase in surface Hydrophilicity in proteins. With increased surface charge in thermostable proteins, researchers have been able to link this to an ability to form networks of sat bridges. This directly contrasts the interaction with water molecules in cold-adapted enzymes. A direct correlation has been found that as ionic interactions become stronger with decreased temperature, a minimization of their number that allows cold-adapted proteins to retain flexibility at low temperatures occurs.

Reference

Siddiqui KS, Cavicchioli R. Cold-Adapted Enzymes. Annual Review of Biochemistry. Vol 75:403-33.Volume publication date July 2006.