A relationship between protein stability and protein function.

Abstract

Enzymes are thought to use their ordered structures to facilitate catalysis. A corollary of this theory suggests that enzyme residues involved in function are not optimized for stability. We tested this hypothesis by mutating functionally important residues in the active site of T4 lysozyme. Six mutations at two catalytic residues, Glu-11 and Asp-20, abolished or reduced enzymatic activity but increased thermal stability by 0.7-1.7 kcal mol-1. Nine mutations at two substrate-binding residues, Ser-117 and Asn-132, increased stability by 1.2-2.0 kcal-mol'1, again at the cost of reduced activity. X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme. In two of these structures the enzyme undergoes a general conformational change, similar to that seen in an enzyme-product complex. These results support a relationship between stability and function for T4 lysozyme. Other evidence suggests that the relationship is general.The ordered, functional structures of proteins reflect two tendencies that are often opposed. On one hand, proteins fold to minimize their free energy. On the other hand, they organize themselves to recognize a ligand or a transition state (1). Minimizing free energy leads to well-packed hydrophobic interiors and hydrophilic exteriors (2). Maximizing function leads to active-site clefts where charged and polar groups are sequestered from water (3, 4) and where hydrophobic patches are exposed to solvent.The hypothesis that there is a balance between stability and function can be stated most strongly as follows: protein residues that contribute to catalysis or ligand binding are not optimal for protein stability. This "stability-function" hypothesis predicts that it usually should be possible to replace residues known to be important for function, reducing protein activity but concomitantly increasing stability of the folded protein.Here we describe experiments that directly test the stabilityfunction hypothesis in T4 lysozyme, an enzyme well characterized for the effects of mutation on structure and stability (5). Five residues were replaced (Table 1 and Fig. 1). These included two residues implicated in chemical catalysis, 11,12), as well as thyee others thought to have a role in substrate binding, Gly-30, Ser-117, and Asp-132. We measured the thermodynamic stability and kinetic activity of the mutant lysozymes. To determine the structural consequences of the substitutions, we determined x-ray crystal structures for several of these proteins. MATERIALS AND METHODSMutagenesis and Protein Purification. Mutations were introduced into the T4 lysozyme gene borne by M13 phage derivative M13mpl8 T4e by mismatched oligonucleotides using the method of Kunkel et al (13) as detailed (6, 14).

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

“…Shoichet et al [38], examining the structure and function of T4 lysozyme, concluded that catalytic residues within the enzyme were not optimized for protein stability and that this was a general property of enzyme active sites. Enzyme active sites, therefore, are more susceptible to unfolding than the rest of the protein molecule due to the increased flexibility required for catalytic function.…”

Role of arginine‐43 and arginine‐69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

“…In addition to co-operativity, the binding-affinity differences could be attributed to the interactions between the catalytic domain of Hin and the DNA. The possibility that Hin residues 43 and 69 are solely involved in catalysis of the DNA inversion reaction, and that inhibition of this reaction stabilizes DNA recognition and binding (Shoichet et al, 1995), cannot be ruled out. Studies with the EcoRV restriction endonuclease suggest that DNAcleavage ability can be linked to DNA binding (Vipond and Halford, 1995).…”

The denaturation and degradation of stable enzymes at high temperatures

“…Too much stability would mean not enough flexibility for effective catalysis, whereas too little stability means too short a useful lifetime [7]. Point mutations which increase enzyme stability often lower specific activity [44,[67][68][69], and vice versa. There is, therefore, strong evidence for the contention that enzyme stability, flexibility and activity are closely interrelated, and that a balance between stabilizing and destabilizing interactions is required to meet the conflicting demands of stability on the one hand, and catalytic function and cellular turnover on the other.…”