Determinants of Enzyme Thermostability Observed in the Molecular Structure ofThermusaquaticusd-Glyceraldehyde-3-phosphate Dehydrogenase at 2.5 Å Resolution^,

Abstract

The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution. To study the determinants of thermostability, we compare our structure to four other GAPDHs. Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed. We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners. These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links. The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds. Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner. Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles. There is, however, an association between thermostability and decreasing enzyme surface to volume ratio. Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs. These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues.

All‐atom contact potential approach to protein thermostability analysis

“…This suggests that these two kinds of contacts may be also important to the thermostability of proteins. This is confirmed to the conclusions of Tanner et al 43 They studied the determinants of thermostability for GAPDH in the extreme thermophile Thermus aquaticus, and found that the number of hydrogen bonds between charged side chains and neutral partners can greatly enhanced the thermostability of proteins. An exception is the thermophilic protein (1xyz) in the 10th family.…”

Structure of Holo-glyceraldehyde-3-phosphate Dehydrogenase fromPalinurus versicolorRefined at 2 Å Resolution

“…The higher average B factor for the coenzyme-binding domain as compared with that of the catalytic domain has been observed previously (Tanner et al, 1996), and this structure shows more obvious differences of the B factors between the two domains. The B factors averaged over the main chain for the two subunits are plotted in Fig.…”

“…GAPDH structures were ®rst determined for the enzymes from lobster (Homarus americanus) muscle, and then for that from the moderate thermophile Bacillus stearothermophilus in several conformational states (Moras et al, 1975;Murthy et al, 1980;Skarzynski et al, 1987;Skarzynski & Wonacott, 1988). In addition, there are several crystallographic investigations for enzymes from other species such as human muscle (Mercer et al, 1976), Bacillus coagulans (Grif®th et al, 1983), Trypanosoma brucei (Vellieux et al, 1993), Thermotoga maritima (Korndorfer et al, 1995), Thermus aquaticus (Tanner et al, 1996), and E. coli (Due  e et al, 1996). These structural investigations provide important information on the folding, catalysis, allosterism mechanism, as well as the thermal stability of this important enzyme.…”

Psychrophilic enzymes: molecular basis of cold adaptation

“…In a recent analysis of the crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase isolated from psychrophiles, mesophiles and thermophiles, the enzyme from the lobster Homarus americanus (which lives at 20°C ) was used as the psychrophilic reference [20]. Although the conclusions of this interesting work are not affected by such a taxonomical error, it is important to reconsider the definition of psychrophily in order to avoid future confusion.…”