Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. 1995 Jan 17; 92(2): 452–456.

A relationship between protein stability and protein function.


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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. [PubMed]
  • Warshel A. Energetics of enzyme catalysis. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5250–5254. [PMC free article] [PubMed]
  • Warshel A, Sussman F, Hwang JK. Evaluation of catalytic free energies in genetically modified proteins. J Mol Biol. 1988 May 5;201(1):139–159. [PubMed]
  • Matthews BW. Structural and genetic analysis of protein stability. Annu Rev Biochem. 1993;62:139–160. [PubMed]
  • Matsumura M, Matthews BW. Control of enzyme activity by an engineered disulfide bond. Science. 1989 Feb 10;243(4892):792–794. [PubMed]
  • Zhang XJ, Baase WA, Matthews BW. Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability. Protein Sci. 1992 Jun;1(6):761–776. [PMC free article] [PubMed]
  • Hardy LW, Poteete AR. Reexamination of the role of Asp20 in catalysis by bacteriophage T4 lysozyme. Biochemistry. 1991 Oct 1;30(39):9457–9463. [PubMed]
  • Anderson DE, Hurley JH, Nicholson H, Baase WA, Matthews BW. Hydrophobic core repacking and aromatic-aromatic interaction in the thermostable mutant of T4 lysozyme Ser 117-->Phe. Protein Sci. 1993 Aug;2(8):1285–1290. [PMC free article] [PubMed]
  • Kuroki R, Weaver LH, Matthews BW. A covalent enzyme-substrate intermediate with saccharide distortion in a mutant T4 lysozyme. Science. 1993 Dec 24;262(5142):2030–2033. [PubMed]
  • Anderson WF, Grütter MG, Remington SJ, Weaver LH, Matthews BW. Crystallographic determination of the mode of binding of oligosaccharides to T4 bacteriophage lysozyme: implications for the mechanism of catalysis. J Mol Biol. 1981 Apr 25;147(4):523–543. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Poteete AR, Sun DP, Nicholson H, Matthews BW. Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft. Biochemistry. 1991 Feb 5;30(5):1425–1432. [PubMed]
  • Dao-Pin S, Baase WA, Matthews BW. A mutant T4 lysozyme (Val 131----Ala) designed to increase thermostability by the reduction of strain within an alpha-helix. Proteins. 1990;7(2):198–204. [PubMed]
  • Becktel WJ, Baase WA. A lysoplate assay for Escherichia coli cell wall-active enzymes. Anal Biochem. 1985 Nov 1;150(2):258–263. [PubMed]
  • Eriksson AE, Baase WA, Matthews BW. Similar hydrophobic replacements of Leu99 and Phe153 within the core of T4 lysozyme have different structural and thermodynamic consequences. J Mol Biol. 1993 Feb 5;229(3):747–769. [PubMed]
  • Tronrud DE. Conjugate-direction minimization: an improved method for the refinement of macromolecules. Acta Crystallogr A. 1992 Nov 1;48(Pt 6):912–916. [PubMed]
  • Jones TA, Zou JY, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. [PubMed]
  • Zhi W, Srere PA, Evans CT. Conformational stability of pig citrate synthase and some active-site mutants. Biochemistry. 1991 Sep 24;30(38):9281–9286. [PubMed]
  • Meiering EM, Serrano L, Fersht AR. Effect of active site residues in barnase on activity and stability. J Mol Biol. 1992 Jun 5;225(3):585–589. [PubMed]
  • Poole LB, Loveys DA, Hale SP, Gerlt JA, Stanczyk SM, Bolton PH. Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability. Biochemistry. 1991 Apr 16;30(15):3621–3627. [PubMed]
  • Hibler DW, Stolowich NJ, Reynolds MA, Gerlt JA, Wilde JA, Bolton PH. Site-directed mutants of staphylococcal nuclease. Detection and localization by 1H NMR spectroscopy of conformational changes accompanying substitutions for glutamic acid-43. Biochemistry. 1987 Sep 22;26(19):6278–6286. [PubMed]
  • Gleason FK. Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function. Protein Sci. 1992 May;1(5):609–616. [PMC free article] [PubMed]
  • Zhang J, Liu ZP, Jones TA, Gierasch LM, Sambrook JF. Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability. Proteins. 1992 Apr;13(2):87–99. [PubMed]
  • Kanzaki H, McPhie P, Miles EW. Effect of single amino acid substitutions at positions 49 and 60 on the thermal unfolding of the tryptophan synthase alpha subunit from Salmonella typhimurium. Arch Biochem Biophys. 1991 Jan;284(1):174–180. [PubMed]
  • Kelley RF, DeVos AM, Cleary S. Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain. Proteins. 1991;11(1):35–44. [PubMed]
  • Kimura S, Nakamura H, Hashimoto T, Oobatake M, Kanaya S. Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart. J Biol Chem. 1992 Oct 25;267(30):21535–21542. [PubMed]
  • Pakula AA, Sauer RT. Amino acid substitutions that increase the thermal stability of the lambda Cro protein. Proteins. 1989;5(3):202–210. [PubMed]
  • Herzberg O, Moult J. Analysis of the steric strain in the polypeptide backbone of protein molecules. Proteins. 1991;11(3):223–229. [PubMed]
  • Williams RJ. The entatic state. Cold Spring Harb Symp Quant Biol. 1972;36:53–62. [PubMed]
  • Mozhaev VV, Berezin IV, Martinek K. Structure-stability relationship in proteins: fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology. CRC Crit Rev Biochem. 1988;23(3):235–281. [PubMed]
  • Varley PG, Pain RH. Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus. J Mol Biol. 1991 Jul 20;220(2):531–538. [PubMed]
  • Eriksson AE, Baase WA, Wozniak JA, Matthews BW. A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. Nature. 1992 Jan 23;355(6358):371–373. [PubMed]
  • Braxton S, Wells JA. Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'. Biochemistry. 1992 Sep 1;31(34):7796–7801. [PubMed]
  • Scrutton NS, Deonarain MP, Berry A, Perham RN. Cooperativity induced by a single mutation at the subunit interface of a dimeric enzyme: glutathione reductase. Science. 1992 Nov 13;258(5085):1140–1143. [PubMed]
  • Inoue M, Yamada H, Hashimoto Y, Yasukochi T, Hamaguchi K, Miki T, Horiuchi T, Imoto T. Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme. Biochemistry. 1992 Sep 22;31(37):8816–8821. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • Protein
    Protein translation features of primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • PubMed
    PubMed citations for these articles
  • Structure
    Three-dimensional structure records in the NCBI Structure database for data reported in the current articles.
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...