Variability in the tertiary structure of α-chymotrypsin at 2.8-Å resolution

Tulinsky, A.; Vandlen, Richard; Morimoto, Carl N.; Mani, N. V.; Wright, L. H.

doi:10.1021/bi00745a023

Cited by 89 publications

(48 citation statements)

References 23 publications

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“…Members ofthis ubiquitous class of proteases hydrolyze peptide bonds and are involved in a broad range of biological processes including intra-and extracellular protein metabolism, digestion, blood coagulation, clot dissolution, immunological response, developmental regulation, and fertilization (1)(2)(3)(4). The three-dimensional structure of serine proteases, in addition to biophysical, molecular biological, and enzymological studies, provides very useful models to understand the mechanism of enzyme action, the basis of substrate specificity, and the molecular evolution of the enzymes themselves (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes.

Sakanari

Staunton

Eakin

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Serine proteases are one of the biologically most important and widely distributed families of enzymes. Isolation of serine protease genes from organisms of widely diverged phylogenetic groups would provide a basis for studying their biological function, the relationship between structure and function, and the molecular evolution of these enzymes. Serine proteases for which little structural information is known are those that are important in the pathogenesis of parasitic nematode and protozoan diseases. Identification and isolation of protease genes from these organisms is a critical first step in understanding their function for the parasite and possibly suggesting innovative approaches to arresting parasitic diseases. Serine protease gene fragments were isolated from genomic DNA of the parasitic nematode Anisakis simplex by using degenerate oligonucleotide primers and the polymerase chain reaction. Primers were designed based upon the consensus sequence of amino acids flanking the active site serine and histidine residues of eukaryotic serine proteases. Four serine protease gene fragments from this parasite were sequenced and one is 67% identical to the rat trypsin II gene. Alignment of these two genes revealed that the intron-exon junctions are conserved between nematode and rat suggesting that this Anisakis serine protease is structurally and functionally similar to rat trypsin. The generality of this approach to identify serine protease genes from genomic DNA of two very divergent species, a parasitic protozoan and a mammal, was also confirmed. Genes for other enzymes or any protein with conserved structural motifs can be identified and isolated using this technology. Using a similar strategy, a cathepsin B-like cysteine (thiol) protease gene fragment was isolated from Caenorhabditis elegans DNA.Serine proteases are one of the most important families of enzymes found in nature. Members ofthis ubiquitous class of proteases hydrolyze peptide bonds and are involved in a broad range of biological processes including intra-and extracellular protein metabolism, digestion, blood coagulation, clot dissolution, immunological response, developmental regulation, and fertilization (1-4). The three-dimensional structure of serine proteases, in addition to biophysical, molecular biological, and enzymological studies, provides very useful models to understand the mechanism of enzyme action, the basis of substrate specificity, and the molecular evolution of the enzymes themselves (5-15).Aside from their role in the physiology and metabolism of organisms, serine proteases have also been implicated in the pathogenesis of a number of infectious diseases. Among the most prevalent of these are parasitic diseases, such as schistosomiasis and onchocerciasis (African river blindness), which

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mentioning

confidence: 99%

Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes.

Sakanari

Staunton

Eakin

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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“…The MR results have established that the native molecule obeys this pseudosymmetry when considered as a rigid body. Small, local deviations could not be detected by this technique or at this resolution, although they are, in fact, anticipated to exist (28). There is no direct evidence for the symmetry of the inhibitor binding.…”

Section: Resultsmentioning

confidence: 70%

“…3). Especially prominent are the a-helices containing residues [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] and 72-88 (in the plane of the sections in Fig. 3) and residues 285-306 and 215-231 (perpendicular to the sections).…”

Section: Resultsmentioning

confidence: 99%

“…There is no direct evidence for the symmetry of the inhibitor binding. The highest inhibitor electron density in the active site pocket is 80% of the maximum density present in the entire map (found in the a-helix containing residues [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], indicating it is unlikely that only one of the noncrystallographically related active sites is occupied. Outside of the binding pocket, however, the inhibitor density is more diffuse, and definite statements concerning the conservation of symmetry cannot be made.…”

Section: Resultsmentioning

confidence: 99%

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Structure of potato inhibitor complex of carboxypeptidase A at 5.5-A resolution.

Rees¹,

Lipscomb²

1980

Proc. Natl. Acad. Sci. U.S.A.

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The complex of the 39-amino acid inhibitor (potato) of bovine carboxypeptidase A (carboxypolypeptidase; peptidyl-Lamino-acid hydrolase, EC 3.4.12.2) was crystallized in space group P32. There are two protein-inhibitor complexes in the asymmetric unit. These crystals exhibited pseudo-P3221 symmetry due to twinning about the a3 axis. Heavy atom difference Patterson maps and rotation functions indicated, however, that the noncrystallographic twofold axis that relates these two complexes is nearly coincident with the a3 axis. Consequently, to a good approximation at low resolution, the space group of the complex is P3221 and the effects of twinning may be ignored. The structure was solved by using multiple isomorphous replacement and molecular replacement techniques. At 5.5-A resolution, the multiple isomorphous replacement map was readily interpretable in terms of the known native carboxypeptidase A structure plus extra density around the active site. The position of this extra density is consistent with the binding mode for extended substrates proposed from earlier model building studies with the native enzyme (Lipscomb, W.

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“…We are involved with a study of the structure of ~-chymotrypsin (~-CHT) at 1.8 ~ resolution (Raghavan & Tulinsky, 1979) with the aim of assessing the structural differences between the two independent molecules of the asymmetric unit (Tulinsky, Vandlen, Morimoto, Mani & Wright, 1973). The phase-extended 1.8 A resolution map of ~-CHT permits an excellent fit of a model to the density.…”

Section: Introductionmentioning

confidence: 99%

A precise and accurate method of measuring Kendrew model coordinates

Frentrup¹,

Tulinsky²

1981

J Appl Cryst

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Polar coordinates of a Kendrew model of ~-chymotrypsin (~-CHT) have been measured with a surveyor's transit and a cathetometer to a high degree of accuracy and precision. Bond distances calculated independently for more than 200 independent aminoacid residues have standard deviations of 0.10 A or less, the torsion angle co has a standard error of 7 °, and the z angle is precise to 3 ° . The measured coordinates of about 1800 non-hydrogen atoms of one molecule of the e-CHT dimer fit a set of idealized polypeptide coordinates with a r.m.s, deviation of 0.17 A, while the average deviation is 0.15 A.

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Variability in the tertiary structure of α-chymotrypsin at 2.8-Å resolution

Cited by 89 publications

References 23 publications

Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes.

Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes.

Structure of potato inhibitor complex of carboxypeptidase A at 5.5-A resolution.

A precise and accurate method of measuring Kendrew model coordinates

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