Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Substrate-assisted catalysis (SAC) is the process by which a functional group in a substrate contributes to catalysis by an enzyme. SAC has been demonstrated for representatives of three major enzyme classes: serine proteases, GTPases, and type II restriction endonucleases, as well as lysozyme and hexose-1-phosphate uridylyltransferase. Moreover, structure-based predictions of SAC have been made for many additional enzymes. Examples of SAC include both naturally occurring enzymes such as type II restriction endonucleases as well as engineered enzymes including serine proteases. In the latter case, a functional group from a substrate can substitute for a catalytic residue replaced by site-directed mutagenesis. From a protein engineering perspective, SAC provides a strategy for drastically changing enzyme substrate specificity or even the reaction catalyzed. From a biological viewpoint, SAC contributes significantly to the activity of some enzymes and may represent a functional intermediate in the evolution of catalysis. This review focuses on advances in engineering enzyme specificity and activity by SAC, together with the biological significance of this phenomenon.
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PMID:Substrate-assisted catalysis: molecular basis and biological significance. 1073 41

The extreme acidothermophilic archaeon Sulfolobus solfataricus harbors a membrane-associated protein kinase activity. Its solubilization and stabilization required detergents, suggesting that this activity resides within an integral membrane protein. The archaeal protein kinase utilized purine nucleotides as phosphoryl donors in vitro. A noticeable preference for nucleotide triphosphates over nucleotide diphosphates and for adenyl nucleotides over the corresponding guanyl ones was observed. The molecular mass of the solubilized, partially purified enzyme was estimated to be approximately 125 kDa by gel filtration chromatography. Catalytic activity resided in a polypeptide with an apparent molecular mass of approximately 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Challenges with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, histone H4, reduced carboxyamidomethylated and maleylated lysozyme, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels in vitro. All of the aforementioned substrates were phosphorylated on threonine residues, while histone H4 was phosphorylated on serine as well. Substitution of serine for the phosphoacceptor threonine in the myosin light chain peptide produced a noticeably inferior substrate. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to a set of known inhibitors of "eukaryotic" protein kinases.
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PMID:The archaeon Sulfolobus solfataricus contains a membrane-associated protein kinase activity that preferentially phosphorylates threonine residues in vitro. 1085 77

One of the major challenges of molecular allergy is to predict the allergenic potential of a protein, particularly in novel foods. Two aspects have to be distinguished: immunogenicity and cross-reactivity. Immunogenicity reflects the potential of a protein to induce IgE antibodies, whereas cross-reactivity is the reactivity of (usually preexisting) IgE antibodies with the target protein. In addition to these two issues, the relation between IgE-binding potential and clinical symptoms is of interest. This is influenced by physical properties (eg, stability and size) and immunologic properties (affinity and epitope valence). Discussions on immunogenicity and cross-reactivity of allergens rely on the establishment of structural similarities and differences among allergens and between allergens and nonallergens. For comparisons between the 3-dimensional protein folds, the representation as 2-dimensional proximity plots provides a convenient visual aid. Analysis of approximately 40 allergenic proteins (or parts of these proteins), of which the protein folds are either known or can be predicted on the basis of homology, indicates that most of these can be classified into 4 structural families: (1) antiparallel beta-strands: the immunoglobulin-fold family (grass group 2, mite group 2), serine proteases (mite group 3, 6, and 9), and soybean-type trypsin inhibitor (Ole e 1, grass group 11); (2) antiparallel beta-sheets intimately associated with one or more alpha-helices: tree group 1, lipocalin, profilin, aspartate protease (cockroach group 2); (3) (alpha+beta) structures, in which the alpha- and beta-structural elements are not intimately associated: mite group 1, lysozyme/lactalbumin, vespid group 5; and (4) alpha-helical: nonspecific lipid transfer protein, seed 2S protein, insect hemoglobin, fish parvalbumin, pollen calmodulin, mellitin from bee venom, Fel d 1 chain 1, serum albumin. Allergens with parallel beta-strands (in combination with an alpha-helix linking the two strands, a motif commonly found in, for example, nucleotide-binding proteins) seem to be underrepresented. The conclusion is that allergens have no characteristic structural features other than that they need to be able to reach (and stimulate) immune cells and mast cells. Within this constraint, any antigen may be allergenic, particularly if it avoids activation of T(H)2-suppressive mechanisms (CD8 cells and T(H)1 cells).
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PMID:Structural biology of allergens. 1093 64

We have extended and applied a multicoordinate free energy method, chemical Monte Carlo/Molecular Dynamics (CMC/MD), to calculate the relative free energies of different amino acid side-chains. CMC/MD allows the calculation of the relative free energies for many chemical species from a single free energy calculation. We have previously shown its utility in host:guest chemistry (Pitera and Kollman, J Am Chem Soc 1998;120:7557-7567)1 and ligand design (Eriksson et al., J Med Chem 1999;42:868-881)2, and here demonstrate its utility in calculations of amino acid properties and protein stability. We first study the relative solvation free energies of N-methylated and acetylated alanine, valine, and serine amino acids. With careful inclusion of rotameric states, internal energies, and both the solution and vacuum states of the calculation, we calculate relative solvation free energies in good agreement with thermodynamic integration (TI) calculations. Interestingly, we find that a significant amount of the unfavorable solvation of valine seen in prior work (Sun et al., J Am Chem Soc 1992;114:6798-6801)3 is caused by restraining the backbone in an extended conformation. In contrast, the solvation free energy of serine is calculated to be less favorable than expected from experiment, due to the formation of a favorable intramolecular hydrogen bond in the vacuum state. These monomer calculations emphasize the need to accurately consider all significant conformations of flexible molecules in free energy calculations. This development of the CMC/MD method paves the way for computations of protein stability analogous to the biochemical technique of "exhaustive mutagenesis." We have carried out just such a calculation at position 133 of T4 lysozyme, where we use CMC/MD to calculate the relative stability of eight different side-chain mutants in a single free energy calculation. Our T4 calculations show good agreement with the prior free energy calculations of Veenstra et al. (Prot Eng 1997;10:789-807)4 and excellent agreement with the experiments of Mendel et al. (Science 1992;256:1798-1802).
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PMID:Exhaustive mutagenesis in silico: multicoordinate free energy calculations on proteins and peptides. 1102 49

Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca(2+) ions. Its activity was optimal at 37 degrees C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.
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PMID:Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization. 1105 22

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.
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PMID:Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus. 1111 74

Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V(L)W92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a peptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fold lower than that of the original antibody. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92 mutant series and changes in the enthalpy of formation. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R(2) = 0.937) as well as increases in the solvent portion of the entropy of binding (R(2) = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistent with values for the hydrophobic effect derived from classical hydrocarbon solubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.
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PMID:Estimation of the hydrophobic effect in an antigen-antibody protein-protein interface. 1111 23

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent-binding sites. Others were intended to displace solvent from preexisting sites. In one case Val-149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild-type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr-152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7 A, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent-binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen-bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen-bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.
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PMID:Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme. 1131 87

Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.
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PMID:Studies on the function of yeast protein disulfide isomerase in renaturation of proteins. 1145 37

A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.
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PMID:Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity. 1153 62


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