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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.
Mol Cell Biol 1986 Jul
PMID:The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases. 353 21

Ab initio quantum mechanical calculations have been used to obtain details of the electron density distribution in a high-resolution refined protein structure. It is shown that with accurate atomic co-ordinates, electron density may be calculated with a quality similar to that which can be obtained directly from crystallographic studies of small organic molecules, and that this density contains information relevant to the understanding of catalysis. Atomic co-ordinates from the 1.8 A and 1.5 A resolution refinements of the crystal structure of protease A from Streptomyces griseus have been used to examine the influence of the environment on the electron density in the side-chain of the active site histidine (His57). The neighbouring aspartic acid 102 is the dominant factor in the environment, and quantum mechanical calculations have been performed on these two residues. Most interesting from the point of view of understanding the catalytic process is the effect that Asp102 has on the electron density in the region of the imidazole nitrogen (N epsilon 2) adjacent to the active site serine 195. In the positively charged imidazolium species, there is a polarization of the N epsilon 2-H bond, reducing the bonding density in a manner that may lower the height of the energy barrier for proton transfer. In the uncharged imidazole species, the proximity of Asp102 causes a movement of density from the lone pair region of the N epsilon 2 into the pi bonding region above and below the plane of the ring. Although it is shown that the primary effect of the aspartic acid is electrostatic, this movement is perpendicular to the direction of the electric field inducing it.
J Mol Biol 1985 Apr 20
PMID:Electron density calculations as an extension of protein structure refinement. Streptomyces griseus protease A at 1.5 A resolution. 389 15

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 A resolution has produced an R index of 0 X 123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O gamma of Ser195 is 1 X 62 A. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.
J Mol Biol 1985 May 05
PMID:The 1.8 A structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates. 389 18

The structure of alpha-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 A resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 A resolution with I greater than or equal to 2 sigma (I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 A. The refined structure was compared with homologous enzymes alpha-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.
J Mol Biol 1985 Aug 05
PMID:Refined structure of alpha-lytic protease at 1.7 A resolution. Analysis of hydrogen bonding and solvent structure. 390 Apr 16

Substrate specificity of two collagenolytic proteases from the king crab Paralithodes camtschatica has been studied. Both proteases are shown to hydrolyze effectively type I and III collagens, gelatin and fibrinogen. The variety of products formed during the enzymatic hydrolysis of the proteins appeared to be different for crab proteases A and C. Studies on peptide hydrolysis demonstrated that protease A cleaves preferably peptide bonds with Arg and Lys as carbonyl components, while protease C prefers hydrophobic amino acids. Kinetic constants of hydrolysis for low molecular weight substrates in the presence of crab proteases have been determined. This allowed us to characterize collagenolytic protease A as a trypsin-like protease. By contrast, collagenolytic protease C was classified as chymotrypsin-like protease although this protease and bovine chymotrypsin are not completely similar. Collagenase substrates Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala were found to be resistant to both crab proteases.
Comp Biochem Physiol Biochem Mol Biol 1994 Mar
PMID:Substrate specificity of collagenolytic proteases from the king crab Paralithodes camtschatica. 774 10

Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER). We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell. Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure. Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP. Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus. When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern. In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization. These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport. Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the "BiP body." This implicates existence of ER subdomains in yeast. A vacuolar protein, proteinase A, appears to colocalize in the BiP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.
Mol Biol Cell 1994 Oct
PMID:Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies. 786 79

We have determined the three-dimensional structure of a proteinase from Streptomyces fradiae ATCC 14544 (SFase-2) at 0.16-nm resolution. SFase-2, a typical serine proteinase, has broad substrate specificity. The characterization and crystallographic analysis of this enzyme have been reported previously [Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T. & Teraoka, H. (1994) Eur. J. Biochem. 220, 55-61]. In the present study, data were collected to approximately 0.16-nm resolution on a Rigaku R-AXIS IIC imaging plate detector system. Preliminary phases were obtained by molecular replacement methods with a search model derived from the previously determined structure of Streptomyces griseus protease A [Sielecki, A. R., Hendrickson, W. A., Broughton, C. G., Delbaere, L. T., Brayer, G. D. & James, M. N. (1979) J. Mol. Biol. 134, 781-804]. The starting model gave an initial crystallographic R factor of 0.443. Refinement with restrained least-squares converged at a final R factor of 0.182 for 16128 observed reflections. The final model includes 86 water molecules. The crystal structure showed that the enzyme consists of two domains, each of which is comprised of a beta barrel with six-stranded beta sheets and two alpha helices. The overall tertiary structure of SFase-2 is similar to the structures of other chymotrypsin-like proteinases from S. griseus, namely proteinase A and proteinase B. The essential residues of the catalytic triad are located on the cleft between the two domains. These two domains have different sequences, but possess similar three-dimensional structures, indicating that a gene duplication event has occurred to produce these two domains. We predicted the tertiary structure of an acidic-amino-acid-specific proteinase on the basis of the crystal structure of SFase-2, and compared the active-site conformations of these two enzymes. We found a characteristic histidine cluster of three histidine residues in the active site of the acidic-amino-acid-specific proteinase. The substrate recognition mechanism of SFase-2 may be mediated through the hydrophobic amino acid residues. However, in the acidic-amino-acid-specific proteinase, the positive charge of this histidine cluster would attract the negative charges of glutamic acid and aspartic acid.
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PMID:Crystal structure analysis of a serine proteinase from Streptomyces fradiae at 0.16-nm resolution and molecular modeling of an acidic-amino-acid-specific proteinase. 792 92

A molecular dynamics (MD) simulation on the contents of two asymmetric units of the crystal structure of the bacterial serine proteinase, Streptomyces griseus proteinase A (SGPA), resulted in four dihydrogen phosphate anions migrating to form a cluster in a solvent region far removed from the protein surface. In an effort to provide experimental verification for this unexpected phenomenon, native SGPA crystals were soaked in 2.0 M KH2AsO4; intensity data were collected and difference electron density maps examined for evidence of H2AsO4- ion clusters. These maps did not corroborate the cluster observed in the MD simulation. They did, however, show positive electron density features located in the active site cavity that could be interpreted as a tetrapeptide, Gly-Ala-Ser-(beta-OH)Asp, covalently bonded to O gamma of Ser195 as an acyl-enzyme intermediate. There was also a peak of electron density corresponding to an ideal position for the hydrolytic water in the deacylation reaction. This density is centred 3.1 A from His57 N epsilon 2 and 2.7 A from the carbonyl-carbon atom of the planar acyl-ester bond of the complex. The carbonyl-oxygen atom of the ester is located in the oxyanion pocket and participates in hydrogen bonds with Gly193 NH (2.6 A) and Ser195 NH (3.0 A).
J Mol Biol 1994 Aug 26
PMID:A crystallographic re-investigation into the structure of Streptomyces griseus proteinase A reveals an acyl-enzyme intermediate. 805 80

The PEP4-encoded aspartate protease proteinase A from Saccharomyces cerevisiae is synthesized as a zymogen (Ammerer, G., Hunter, C. P., Rothman, J. H., Saari, G. C., Valls, L. A., and Stevens, T. H. (1986) Mol. Cell. Biol. 6, 2490-2499; Woolford, C. A., Daniels, L. B., Park, F. J., Jones, E. W., van Arsdell, J. N., and Innis, M. A. (1986) Mol. Cell. Biol. 6, 2500-2510). We constructed a mutant form, lacking the propeptide. This form, still containing the signal peptide, was translocated into the endoplasmic reticulum, but the mature region was subsequently completely degraded. When a plasmid encoding only the propeptide after the signal peptide was introduced into a strain producing the mature region, a subpopulation of mature region molecules was rescued from the degradation and gained activity. Increased activity was found when the mature region was co-produced with increased amounts of propeptide, whereas truncated propeptides, lacking residues at its C terminus, were less efficient in the interaction with the mature region. We propose that the mature region of proteinase A cannot fold into its stable active conformation in the absence of the propeptide and that the propeptide can promote folding of the mature region, even when the propeptide and the mature region are not covalently linked.
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PMID:The propeptide is required for in vivo formation of stable active yeast proteinase A and can function even when not covalently linked to the mature region. 834 80

Identification of Na+ binding sites in protein crystals is complicated by comparable electron density of this monovalent cation and water. Valence calculations can predict the location of metal ion binding sites in proteins with high precision. These calculations were used to screen 332,242 water molecules in 2742 protein structures reported in the Protein Data Bank (PDB), searching for molecules with Na+/- specific valence values V(Na+) > or = 1.0 v.u., as expected for a bound Na ion. Thirty-three water molecules (<0.01% of the total) were found be have V(Na+) > or = 1.0 v.u. and to be located within 3.5 A from at least two protein oxygen atoms. These water molecules, with a high Na+ -specific valence, do not have valences specific for other cations, like Li+, K+, Mg2+ or Ca2+. They belong to nine different proteins (deoxyribonuclease I, enolase, hen egg-white lysozyme, human lysozyme, phospholipase A2, proteinase A, rubredoxin, thrombin and phage T4 lysozyme) and appear with similar coordination geometry, typically octahedral, in the same place in multiple crystal structure determinations of the same protein. In the case of thrombin, the water molecule singled out by valence calculations is, in fact, a bound Na ion as demonstrated by molecular replacement with Rb+. Valence calculations provide an accurate screening of water in protein crystals and may help identify Na+ binding sites of functional importance.
J Mol Biol 1996 Feb 23
PMID:Valence screening of water in protein crystals reveals potential Na+ binding sites. 859 92


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