Case 5
Characterization of Subtilisin from the Antarctic Psychrophile Bacillus TA41
Last modified 17 January 2005
Focus concept
The structural features involved in protein adaptation to cold temperatures are explored.
Prerequisites
Protein architecture.
Principles of protein folding.
Background
Although most organisms live at a temperature of 37C, some organisms have the ability to survive at extreme temperatures of heat and cold. Bacteria referred to as thermophiles can survive in hot springs at temperatures up to 120C. Other organisms such as bacteria found in Antarctic seawater are capable of surviving at temperatures below 0C and are referred to as psychrophiles. Growth at such extreme temperatures requires adaptation of the organism’s proteins so that enzymatic reactions will still be able to take place. For the thermophiles, a greater level of stability is required so that the proteins will not denature at the high temperature. This stability is achieved through ion pairs, hydrophobic interactions, and ion binding. In addition, thermophilic proteins tend to be compact because they have fewer hydrophilic amino acid residues which have a preference for interacting with the solvent. Adaptation at cold temperatures is slightly different because additional stability is not needed. Instead, because reaction rates typically decrease with decreasing temperature, psychrophilic enzymes must compensate by being able to accommodate their substrates easily so that the reaction can take place in a timely manner. This requires proteins with additional conformational flexibility and decreased stability.
The properties of thermophilic proteins have been extensively studied, but not as much is known about their psychrophilic counterparts. In this case, the investigators purified and characterized the protease subtilisin S41 from the Antarctic psychrophile Bacillus TA41. Subtilisin was chosen because the properties of nearly 50 subtilisin-like proteins are known. Subtilisin S41 is secreted by the Bacillus into its growth medium, making isolation of this enzyme a fairly straightforward process. Once purified, the subtilisin S41 was sequenced and characterized. The activity of the subtilisin protease enzyme was measured by adding the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF) and measuring the absorbance of the yellow p-nitroaniline product at 412 nm. The reaction is shown in Figure 5.1. The properties of the psychrophilic subtilisin were then compared with subtilisin from mesophiles, organisms that grow at more moderate temperatures.
Understanding the factors that allow a protein to adapt to a cold environment may provide clues to the protein folding process.
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Figure 5.1: Enzymatic assay for subtilisin activity.
Questions
1. Following purification of the subtilisin S41 enzyme, the investigators carried out analytical procedures to determine the protein’s molecular weight and pI.
a. What technique would they have used to determine the protein’s molecular weight? What technique would they have used to determine the protein’s pI?
b. The pI of the psychrophilic subtilisin S41 enzyme was determined to be 5.3. This value is much lower than the pI of mesophilic subtilisins, which have pI values ranging from 9-11. Which amino acid residues does the subtilisin S41 have in greater amounts than the mesophilic subtilisins?
2. The investigators then carried out an experiment in which they incubated subtilisin S41 and subtilisin Carlsberg (a mesophilic subtilisin) at 50C and measured enzymatic activity as a function of time. The results are shown in Table 5.1. Interpret these results and explain their significance.
Table 5.1: Thermal stability of subtilisin S41 and subtilisin Carlsberg. Enzymes were incubated at 50C and enzyme activity was measured at periodic intervals. The half-times of inactivation, ti, (time required to cause a 50% reduction in enzyme activity) are reported, as well as the optimum temperature of activity.
Subtilisin S41 / 3 / 40
Subtilisin Carlsberg / 24 / 60
3. The properties of psychrophilic, mesophilic, and thermophilic subtilisin enzymes were compared and the results are shown in Table 5.2. Using these data and data in Question 2, describe the general characteristics of psychrophilic subtilisin, and compare these characteristics with the mesophilic and thermophilic subtilisins.
Table 5.2: Main structural features of subtilisins are compared. (Based on Davail, et al., 1994.)
Psychrophilic / Mesophilic / ThermophilicS41 / BPN / Carlsberg / Savinase / Thermitase
Amino Acids / 309 / 275 / 274 / 269 / 279
Asp Content / 21 / 10 / 9 / 5 / 13
Ionic Interactions / 2 / 5 / 3 / 7 / 10
Aromatic Interactions / 0 / 5 / 3 / 7 / 10
Kd for Ca2+ (M)* / 10-6 M / 10-10 M / 10-10 M / not known / < 10-10 M
* The Kd value is the Ca2+ concentration required to achieve 50% calcium binding to the subtilisin.
Reference
Davail, S., Feller, G., Narinx, E., and Gerday, C. (1994) J. Biol.Chem., 269, pp. 17448-17453.
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