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Biomedical subjects

M Vihinen

Publications and source records attributed to M Vihinen.

15 recordsLinked to original sources
PubMed1992

Human aspartylglucosaminidase. A biochemical and immunocytochemical characterization of the enzyme in normal and aspartylglucosaminuria fibroblasts.

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is an essential enzyme in the degradation of asparagine-linked glycoproteins. In man, deficient activity of this enzyme leads to aspartylglucosaminuria (AGU), a recessively inherited lysosomal storage disease. Here we used affinity-purified polyclonal antibodies against the native AGA and its denatured subunits to establish the molecular structure and intracellular location of the enzyme in normal and AGU fibroblasts. Inactivation of the enzyme was found to coincide with the dissociation of the heterodimeric enzyme complex into subunits. Although the subunits were not linked by covalent forces, the intrapolypeptide disulphide bridges were found to be essential for the normal function of AGA. AGA was localized into lysosomes in control fibroblasts by both immunofluorescence microscopy and immuno-electron microscopy, whereas in AGU cells the location of antigen was different, suggesting that, owing to the mutation, a missing disulphide bridge, most of the enzyme molecules get retarded in the cis-Golgi region and most probably face intracellular degradation.

Aspartylglucosaminuria↗
PubMed1992

Computer modeling of two inorganic pyrophosphatases.

The yeast Saccharomyces cerevisiae has two inorganic pyrophosphatases that are structurally related. One, PPA1, is a cytoplasmic enzyme. The other, PPA2, is located in the mitochondria and appears to be energy-linked. The sequence similarity of PPA1 and PPA2 is about 66% and the identity is about 50%. All amino acids known to be important for catalysis are conserved, except one glutamate which is substituted by an aspartate in PPA2. The structures of PPA2 and the cytoplasmic PPase from Schizosaccharomyces pombe were modeled based on the three dimensional structure of PPA1. Two cysteines in PPA2 and one in the S. pombe enzyme are located at the catalytic cleft. Four residues form an unique insertion near the entrance of the catalytic cleft in the mitochondrial enzyme.

Amino Acid Sequence↗
PubMed1992

Selection of a representative set of structures from Brookhaven Protein Data Bank.

Reliable structural and statistical analyses of three dimensional protein structures should be based on unbiased data. The Protein Data Bank is highly redundant, containing several entries for identical or very similar sequences. A technique was developed for clustering the known structures based on their sequences and contents of alpha- and beta-structures. First, sequences were aligned pairwise. A representative sample of sequences was then obtained by grouping similar sequences together, and selecting a typical representative from each group. The similarity significance threshold needed in the clustering method was found by analyzing similarities of random sequences. Because three dimensional structures for proteins of same structural class are generally more conserved than their sequences, the proteins were clustered also according to their contents of secondary structural elements. The results of these clusterings indicate conservation of alpha- and beta-structures even when sequence similarity is relatively low. An unbiased sample of 103 high resolution structures, representing a wide variety of proteins, was chosen based on the suggestions made by the clustering algorithm. The proteins were divided into structural classes according to their contents and ratios of secondary structural elements. Previous classifications have suffered from subjective view of secondary structures, whereas here the classification was based on backbone geometry. The concise view lead to reclassification of some structures. The representative set of structures facilitates unbiased analyses of relationships between protein sequence, function, and structure as well as of structural characteristics.

Algorithms↗
PubMed1992

MULTICOMP: a program package for multiple sequence comparison.

An algorithm for multiple sequence comparison was implemented in FORTRAN 77 for VAX/VMS in GCG-compatible format. The MULTICOMP program package includes several procedures with which one query sequence can be compared simultaneously to several DNA, RNA or amino acid sequences. The same technique was also introduced for comparing propensities of secondary structural features, which can be predicted on the basis of amino acid sequences. The technique has been applied to a wide range of sequence and structural analyses.

Algorithms↗
PubMed1991

Amino acid sequence similarities of the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase and peroxisomal multifunctional delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase enzyme in rat liver. The proposed occurrence of isomerization and hydration in the same catalytic domain of the multifunctional enzyme.

We report the isolation and characterization of a cDNA encoding the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase from rat liver. Tryptic fragments of the purified protein were generated, purified, and sequenced. A rat liver cDNA library, constructed in the plasmid vector pUEX1 was screened with oligonucleotides synthesized on the basis of peptide sequences. The obtained clone contained 783 bases predicting to code the entire mature protein of 261 amino acids. The molecular weight of 29,300 predicted from cDNA-derived sequences was consistent with the subunit size determined earlier. A high degree of similarity was noted between the amino acid sequence of isomerase and that of the amino-terminal half of peroxisomal multifunctional isomerase-hydratase-dehydrogenase enzyme and mitochondrial 2-enoyl-CoA hydratase in rat liver. These similarities also appeared at the level of predicted secondary structural elements, suggesting that hte rat multifunctional enzyme has both the isomerization and hydration activities in the amino-terminal domain. This idea is further supported by the proposed existence of only one CoA-binding site in the amino-terminal half of the multifunctional enzyme and by previous studies suggesting that the transfer of the substrate from the isomerization site to the hydration site occurs without aqueous bulk phase (Palosaari P.M., and Hiltunen, J. K. (1990) J. Biol. Chem. 265, 2446-2449).

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1990

Conservation of functional residues between yeast and E. coli inorganic pyrophosphatases.

The alignments of the amino acid sequences of inorganic pyrophosphatase (PPase) from Saccharomyces cerevisiae (Y1-PPase, 286 amino acids) and Escherichia coli (E-PPase, 175 amino acids) are examined in the light of crystallographic and chemical modification results placing specific amino acid residues at the active site of the yeast enzyme. The major results are: (1) the full E-PPase sequence aligns within residues 28-225 of Y1-PPase, raising the possibility that the N-terminal and C-terminal portions of Y1-PPase may not be essential for activity, and (2) that whereas the overall identity between the two sequences is only modest (22-27% depending on the choice of alignment parameters), of some 17 putative active site residues, 14-16 are identical between Y-PPase and E-PPase. PPase thus appears to be an example of enzymes from widely divergent species that conserve common functional elements within the context of substantial overall sequence variation.

Amino Acid Sequence↗
PubMed1990

Conserved residues of liquefying alpha-amylases are concentrated in the vicinity of active site.

Three dimensional structure of three liquefying type Bacillus alpha-amylases were modeled based on sequence analyses and refined structure of Aspergillus oryzae enzyme. The models suggest that the overall folding motif of alpha-amylases is conserved. The active site, substrate binding and stabilizing calcium binding residues are conserved and concentrated in a cleft between two domains. They constitute the core of alpha-amylases to which other, less conserved regions are attached. The bacterial enzymes have a loop of about 45 residues near the active site and Ca2+ binding region. The loop may be important for the liquefying function of these enzymes.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.

Amino Acid Sequence↗
PubMed1990

Characterization of a thermostable Bacillus stearothermophilus alpha-amylase.

Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.

Amino Acid Sequence↗
PubMed1989

Microbial amylolytic enzymes.

Starch-degrading, amylolytic enzymes are widely distributed among microbes. Several activities are required to hydrolyze starch to its glucose units. These enzymes include alpha-amylase, beta-amylase, glucoamylase, alpha-glucosidase, pullulan-degrading enzymes, exoacting enzymes yielding alpha-type endproducts, and cyclodextrin glycosyltransferase. Properties of these enzymes vary and are somewhat linked to the environmental circumstances of the producing organisms. Features of the enzymes, their action patterns, physicochemical properties, occurrence, genetics, and results obtained from cloning of the genes are described. Among all the amylolytic enzymes, the genetics of alpha-amylase in Bacillus subtilis are best known. Alpha-Amylase production in B. subtilis is regulated by several genetic elements, many of which have synergistic effects. Genes encoding enzymes from all the amylolytic enzyme groups dealt with here have been cloned, and the sequences have been found to contain some highly conserved regions thought to be essential for their action and/or structure. Glucoamylase appears usually in several forms, which seem to be the results of a variety of mechanisms, including heterogeneous glycosylation, limited proteolysis, multiple modes of mRNA splicing, and the presence of several structural genes.

Bacteria↗
PubMed1988

An algorithm for simultaneous comparison of several sequences.

An algorithm was developed to compare simultaneously several DNA, RNA or protein sequences. With the algorithm, conserved regions of one sequence are located by doing pairwise comparisons with other sequences, which is advantageous in planning site-directed mutagenesis studies. The observation matrices filled with scores of comparisons are superimposed and added together and those points having values greater than or equal to stringency are accepted. The predicted secondary structural features can also be compared.

Algorithms↗
PubMed1987

Relationship of protein flexibility to thermostability.

Thermostability of proteins arises from the simultaneous effect of several forces, which in fact lead to decreased flexibility of the polypeptide chain. This is verified by flexibility indices, which are derived from normalized B-values of individual amino acids in several refined three-dimensional structures. Flexibility indices show that overall flexibility is reduced when thermostability is increased. Protein molecules require both flexibility and rigidity to function, but the higher the temperature optimum and stability the more rigid is the structure needed to compensate for increased thermal fluctuations. Flexibilities of proteins performing the same catalytic activity seem to be about the same at their temperature optima, but the more rigid thermostable proteins reach the flexibility of thermolabile proteins at higher temperatures. In several proteins such as allosteric enzymes, some local sites of flexibility are highly conserved. The relevance of reduced flexibility to overall stability of proteins is also discussed. Flexibility indices and profiles can be used in the design of more stable proteins by site-directed mutagenesis.

Mutation↗