Replacement of His12 or His119 of bovine pancreatic ribonuclease A with acidic amino acid residues for the modification of activity and stability.

In an attempt to produce a bovine pancreatic ribonuclease A (RNase A) with increased activity and stability, the catalytic pair of His12 and His119 was substituted with aspartic acid or glutamic acid, and aspartic acid, respectively, to evaluate the role of the two histidine residues in the activity and stability. Kinetic analysis revealed that k(cat)/K(m) values were significantly reduced for all mutant enzymes due to a decreased k(cat) rather than an increased K(m): the k(cat) values for both CpA and C>p of H12D and H12E decreased to about 1/1000; the k(cat) values of H119D decreased by 1/3300 for CpA and 1/80 for C>p. Thus, neither Asp nor Glu is able to act solely as an efficient catalytic residue of RNase A.

Crystallization and preliminary X-ray analysis of carboxypeptidase Y inhibitor IC complexed with the cognate proteinase.

Carboxypeptidase Y (CPY) inhibitor I(C) is a naturally occurring serine carboxypeptidase inhibitor from Saccharomyces cerevisiae, the sequence of which is not homologous with any other known proteinase inhibitor and is classified as the phosphatidylethanolamine-binding protein (PEBP). I(C) has been crystallized in complex with the deglycosylated form of CPY by the hanging-drop vapour-diffusion technique with ammonium sulfate as a precipitant. The crystals of the complex belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 81.

Indiscriminate glycosylation of procarboxypeptidase Y expressed in Pichia pastoris.

To obtain large amounts of deglycosylated procarboxypeptidase Y (proCPY), in which all of the N-glycosylation sites were replaced by alanine residue by the point mutation method, an expression system was constructed using Pichia pastoris. The secreted enzyme was characterized by SDS-PAGE, native PAGE, MALDI-TOF mass spectrometry, and dynamic light scattering, and the results indicated heterogeneity. The recombinant proCPY contained 29 mol of glucose per mole of protein in average, according to the carbohydrate analysis by the phenol-sulfuric acid method.

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure.

The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety.

Minimization of cavity size ensures protein stability and folding: structures of Phe46-replaced bovine pancreatic RNase A.

The Phe46 residue, located in the hydrophobic core of RNase A, was replaced with other hydrophobic residues, leucine, valine, or alanine, and their X-ray crystallographic structures were determined up to 1.50-1.80 A resolution in an attempt to examine the relationship between structural changes and conformational stability or folding kinetics.

The multiple site binding of carboxypeptidase Y inhibitor (IC) to the cognate proteinase. Implications for the biological roles of the phosphatidylethanolamine-binding protein.

The serine carboxypeptidase inhibitor in the cytoplasm of Saccharomyces cerevisiae, IC, specifically inhibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs). In the presence of 1 M guanidine hydrochloride, a CPY-IC complex is formed and is almost fully activated. The reactivities of phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid, and diisopropyl fluorophosphate toward the complex are considerably increased in 1 M guanidine hydrochloride, indicating that IC contains a binding site other than its inhibitory reactive site.

Overexpression and functional characterization of a serine carboxypeptidase inhibitor (I(C)) from Saccharomyces cerevisiae.

Carboxypeptidase Y (CPY) inhibitor, I(C), a cytoplasmic inhibitor of vacuolar proteinases in yeast, Saccharomyces cerevisiae, was purified by means of a high-level expression system using a proteinase-deficient strain, BJ2168, and an expression vector with the promoter GAL1. The purified I(C) exists as a monomeric beta-protein in solution with a mole-cular weight of 24,398.4 as determined by gel filtration chromatography, MALDI-TOF mass spectrometry, and far-UV CD spectroscopy.

N-terminal acetyl group is essential for the inhibitory function of carboxypeptidase Y inhibitor (I(C)).

Carboxypeptidase Y (CPY) inhibitor, I(C), a yeast cytoplasmic inhibitor in which the N-terminal amino acid is acetylated, was expressed in Escherichia coli and produced as an unacetylated form of I(C) (unaI(C)). Circular dichroism and fluorescence measurements showed that unaI(C) and I(C) were structurally identical and produce identical complexes with CPY. However, the K(i) values for unaI(C) for anilidase and peptidase activity of CPY were much larger, by 700- and 60-fold, respectively, than those of I(C).

Sub-zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure.

High hydrostatic pressure induced cold inactivation of carboxypeptidase Y. Carboxypeptidase Y was fully active when exposed to subzero temperature at 0.1 MPa; however, the enzyme became inactive when high hydrostatic pressure and subzero temperature were both applied.

Increased proteolytic susceptibility of carboxypeptidase Y caused by modification of the disulfide zipper.

To investigate the structural importance of a "disulfide zipper" motif of carboxypeptidase Y, disulfide-deficient mutant enzymes were expressed in two strains of Saccharomyces cerevisiae. The mutant enzymes were rapidly degraded into fragments by intracellular proteases. Thus, it is concluded that the disulfide zipper is essential in maintaining the structural integrity of CPase Y against proteolytic susceptibility.

Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y.

Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket.

High pressure in bioscience and biotechnology: pure science encompassed in pursuit of value.

A fundamental factors, pressure (P), is indispensable to develop and support applications in the field of bioscience and biotechnology. This short sentence describes an example how high pressure bioscience and biotechnology, which started from applied science, stimulates challenges of basic science and pure science in the biology-related fields including not only food science, medicine, and pharmacology but also biochemistry, molecular biology, cell biology, physical chemistry, and engineering.

Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site.

To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V.

Significance of the four carboxyl terminal amino acid residues of bovine pancreatic ribonuclease A for structural folding.

The C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) form a core structure in the initial stage of the folding process that leads to the formation of the tertiary structure. In this paper, roles of the C-terminal four amino acids in the structure, function, and refolding were studied by use of recombinant mutant enzymes in which these residues were deleted or replaced. Purified mutant enzymes were analyzed for their secondary structure, thermal stability, and ability to regenerate from the denatured and reduced state.

High pressure-induced changes of biological membrane. Study on the membrane-bound Na(+)/K(+)-ATPase as a model system.

In order to study the pressure-induced changes of biological membrane, hydrostatic pressures of from 0.1 to 400 MPa were applied to membrane-bound Na(+)/K(+)-ATPase from pig kidney as a model system of protein and lipid membrane. The activity showed at least a three-step change induced by pressures of 0.

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 A resolution.

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants--F120A, F120G, and F120W--were analyzed up to a 1.4 A resolution.