Mutant Lysozymes Research Articles

Amino acid substitutions far from the active site of bacteriophage T4 lysozyme reduce catalytic activity and suggest that the C-terminal Lobe of the enzyme participates in substrate binding

As part of a systematic study of the effect of single amino acid substitutions on the structure, function and stability of the lysozyme from bacteriophage T4, we have characterized a mutant lysozyme for which the catalytic activity is reduced to 4% that of the wild-type enzyme, yet the location of the amino acid substitution is on the far side of the molecule, 25 A from the active site. Genetic and chemical analysis show that the mutant lysozyme differs from wild-type only in the replacement of Glu128 by a lysine residue. Crystallographic analysis confirms the location of the amino acid substitution, and also shows that the replacement of Glu128, which is on the surface of the protein, causes very little change in the three-dimensional structure of the lysozyme molecule. Thermodynamic analysis shows that the stability of the mutant lysozyme is comparable with that of native lysozyme. These results suggest that Glu128 participates directly in substrate binding and/or catalysis, and argue against the possibility that the low activity of the mutant enzyme is due to an indirect effect such as destabilization of the lysozyme molecule, or modification of its three-dimensional structure. Glu128 is located in the part of the carboxy-terminal domain of phage lysozyme that has no counterpart in the structure of hen egg-white lysozyme. We suggest that the role of this C-terminal domain is to bind the peptide cross-link that connects neighboring saccharide strands within the cell walls of Escherichia coli . This postulate is consistent with the known differences in specificity between phage lysozyme and hen egg-white lysozyme, and is also compatible with the activity of native and mutant phage lysozymes toward different bacterial cell walls.

Mutations and protein stability.

AbstractThe comparative study of proteins which differ in primary structure by point mutations permits one to use thermodynamic experiments to obtain information about the role of specific amino acids in determining protein structure and stability. We have now determined the thermodynamic changes induced in six mutants of T4 lysozyme and have compared the results with the wildtype enzyme. Our work is in collaboration with B. Matthews and his colleagues, who have determined the crystal structure of T4 lysozyme and have obtained difference Fourier maps for four of the mutants. The ultimate aim is to correlate changes in protein stability with changes in the detailed structure of the protein. This paper discusses the thermodynamic results obtained from the mutants studied. All the mutants have a lower Tm than the wild‐type enzyme and changes in the enthalpy of denaturation are sometimes extraordinarily large. Changes in ΔH of denaturation are usually accompanied by compensating changes in ΔS. The general question of protein stability and the manner in which it varies with temperature and mutations is discussed.

Structure of the lysozyme from bacteriophage T4: An electron density map at 2.4 Å resolution

The three-dimensional structure of the lysozyme from bacteriophage T4 has been determined from a 2.4 Å resolution electron density map. The map, determined using isomorphous replacement and anomalous scattering differences from platinum and mercury-substituted crystals, reveals the approximate conformation of the molecule unambiguously. Details of the structure determination are presented and the conformation of the molecule is described in detail. The observed properties of those mutant lysozymes for which the changes in the amino acid sequence have been determined are discussed in the light of the three-dimensional structure. In most cases the behavior of the mutant lysozymes can be easily rationalized, but there are a few instances where this is not so.

Stability of phage T4 lysozymes I. Native properties and thermal stability of wild type and two mutant lysozymes

Two mutants of phage T4 lysozyme were prepared and characterized. One mutation substituted a tyrosine residue for tryptophan at position 138. The other substituted tyrosines at all three tryptophan positions of the wild type molecule (126, 138, 158). Comparative studies of the physical properties (absorption, flourescence, circular dichroism) of the three enzymes were performed as a function of pH. Also, the proteins were reversibly melted as a function of pH. Since the unfolding reaction appeared to be a two-state process for all these proteins, the data were analyzed by the van 't Hoff procedure. The changes in stability and activity produced by substitution of Trp 138 were especially significant. The other substitutions were neutral. See the end of the paper for a summary of conclusions. In the appendix the appropriate thermodynamic relations are developed for a constant ΔC P transition. Parts of this material were presented at the meeting of the Society of Biological Chemists in San Francisco in June, 1976 and at the International Conference on Biomolecules at Regensburg, G.F.R. in October, 1976.

Effect of neighboring nucleotide sequences on suppression efficiency in amber mutants of T4 phage lysozyme.

Variations in suppression efficiency were observed among nonsense mutations at different locations within the lysozyme gene (e) of T4 phage. The present experiments using three amber mutants in lysozyme gene indicate such variations presumably depend upon the base sequences neighboring to the nonsense mutations.

Bacteriophage T7 Lysozyme Is an N-Acetylmuramyl-l-alanine Amidase

Abstract Studies on the specificity of a lytic enzyme induced by bacteriophage T7 infection of Escherichia coli were carried out by analyzing the products of the enzyme's action on purified E. coli peptidoglycan. The E. coli peptidoglycan is known to contain a lipoprotein (molecular weight = 7,200) covalently attached to diaminopimelic acid. When radioactively labeled E. coli peptidoglycan was digested with purified bacteriophage T4 lysozyme, the lipoprotein was cleaved from the peptidoglycan and migrated to a position of molecular weight 10,600 in a sodium dodecyl sulfate polyacrylamide gel electrophoretogram. The lipoprotein was found to contain both radioactive N-acetylglucosamine and diaminopimelic acid, as expected from the known enzymatic specificity of T4 lysozyme (an N-acetylmuramidase). On the other hand, when the labeled peptidoglycan was digested with a bacteriophage T7 lysate, the lipoprotein migrated to a position of significantly lower molecular weight than that of the lipoprotein cleaved by T4 lysozyme. Furthermore, the lipoprotein contained diaminopimelic acid, but not N-acetylglucosamine. This suggested that the release of the lipoprotein from the peptidoglycan by the T7 lysate was not due to the hydrolysis of one of the bonds in the glycan backbone, but due to the hydrolysis of one of the bonds in the peptide portion of the peptidoglycan. Peptidoglycan radioactively labeled with l-alanine was prepared, digested with the lytic enzyme in a T7 lysate, and the digestion products analyzed. From these experiments, it was concluded that the T7 enzyme cleaves the bond between N-acetylmuramic acid and l-alanine. This conclusion was confirmed by observing that the release of NH2-terminal l-[14C]alanine was chemically detected by dinitrophenylation after digestion of labeled peptidoglycan with the T7 lysate. Hence the T7 lytic enzyme is an N-acetylmuramyl-l-alanine amidase. It is most likely the product of the T7 lysozyme gene (gene 3.5), since no lytic activity was detected in a lysate prepared by infection of E. coli with a T7 lysozyme mutant.

Frameshift mutation in the lysozyme gene of bacteriophage T4: Demonstration of the insertion of five bases, and a summary of in vivo codons and lysozyme activities

Two pseudo-wild type mutants carrying double frameshifts, eJR13 eJD5 and eJR14 eJD5, in the lysozyme gene of bacteriophage T4 were obtained from a single mutant, eJD5, by reversion with proflavin, and the primary structures of these lysozymes were studied. It was found that the amino acid sequence 20 Asp-Thr-Glu in the wild-type strain was altered to Glu-Thr-Gln and Asp-Lys-Thr-Gln in eJR14 eJD5 and eJR13 eJD5, respectively, The mutation in eJD5 was previously identified as the deletion of two adjacent bases. From this fact, the above changes of amino acid sequences in eJR13- eJD5 and eJR14 eJD5 could be explained by the addition of five and two bases in the loci of the mutations of eJR13 and eJR14 respectively. The addition of five bases caused by a single-step frameshift mutation has not hither to been reported, although the additions and deletions of one base, two bases, and addition only of four bases had been found previously. The codons GAC (aspartic acid) in the wild-type strain, and CAA (glutamine) and ACA (threonine) in the mutants were identified as being utilized in vivo. By summarizing our results and previously published data, close correlation between the bases at the third position in codon triplets and the base ratio of DNA is deduced. The enzyme activities of the present two mutants were found to be markedly low. The twentieth aspartic acid and some amino acid(s) in the neighbouring region seem to have an important role in the enzyme activity. Further, the effects on the enzyme activity of additions and/or alterations of amino acid(s) in various mutant lysozymes are discussed.