Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins

Abstract

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17 degreesC higher melting temperature. Its temperature optimum is 22-25 degreesC higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a "corresponding state" regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

Figure 1

Specific activity-temperature profiles of E. coli (x) and Th. thermophilus (+) IPMDHs, in 0.3 M and 1.0 M KCl, respectively, in 20 mM potassium phosphate buffer pH 7.6 containing 0.2 mM MnCl₂.

Figure 2

Calorimetric melting profile of E. coli (thin line) and Th. thermophilus (thick line) IPMDHs measured with a heating rate of 1°C/min, in 20 mM potassium phosphate, pH 7.6. KCl concentrations were set to 0.3 M and 1.0 M, respectively. Melting temperatures are 73.5°C for the E. coli and 90.0°C for the Th. thermophilus enzymes.

Figure 3

Heat denaturation curves of E. coli (thin line) and Th. thermophilus (thick line) IPMDHs in D₂O followed by CD with a heating rate of 50°C/h at 221 nm in 20 mM potassium phosphate buffer containing 0.3 M or 1.0 M KCl. Measurements were carried out at pD 7.15 (A) and pD 8.15 (B). Corresponding melting temperatures are 77.4°C for the E. coli and 92.2°C for the Th. thermophilus enzyme (pD 7.15) and 74.5°C and 90.9°C (pD 8.15).

Figure 4

A typical H/D exchange experiment on E. coli IPMDH at 25°C, pD 7.15 in the time range from 30 sec to 24 h. The amide II band (at approximately 1,550 cm⁻¹) shows the decreasing number of amide protons. The broad band at 1,450 cm⁻¹ reflects the increasing number of ND groups and HDO molecules. Arrows show the direction of changes. Spectra of undeuterated and totally deuterated proteins (thin lines) were measured as described in Materials and Methods.

Figure 5

H/D exchange data, summarized in the form of relaxation spectra for both E. coli and Th. thermophilus IPMDHs at 25°C. E. coli, pD 7.15 (▵), pD 8.15 (□); Th. thermophilus, pD 7.15 (▿), pD 8.15 (○). X is the fraction of unexchanged peptide hydrogens, t is the time. k₀, the chemical exchange rate constant, was calculated according to Eq. 4. The solid lines represent the exchange rate curves for hypothetical polypeptides characterized by the ρ values indicated in the figure. ρ is the probability of solvent exposure of the peptide groups (see Materials and Methods). Curves indicate a more rigid structure for the thermophilic enzyme.

Figure 6

H/D exchange data of IPMDHs, at two different pD values at their temperature optima. E. coli, 48°C, pD 7.15 (▵), pD 8.15 (□); Th. thermophilus, 70°C, pD 7.15 (▿), pD 8.15 (○). Curves obtained for the two enzymes show very similar flexibilities.