Biomedical subjects

K Y Choi

Publications and source records attributed to K Y Choi.

At least 73 records · Page 4Linked to original sources

PubMed1997

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.

Aspartic Acid↗

PubMed1997

Recent changes in the pattern of hand injuries in Hong Kong: a regional hospital survey.

Adult inpatients with injuries to the hand admitted to the Department of Orthopaedics and Traumatology of the Prince of Wales Hospital, Hong Kong, from June 1992 to May 1993 were surveyed (n=533). The causes of injury, mechanisms involved, and the nature of injuries were recorded. The injuries were further graded according to four different scales. It was found that 65% of the injuries were sustained at work, 15% in domestic environments, 7.6% during sports or recreational activities, and 6.5% in traffic accidents. Injuries caused by heavy machinery amounted to 26.7% and the electric saw was the second most common cause of injury accounting for 9.4% of injuries. Crush injuries occurred in 37.8%. The incidence of hand injury is estimated to be 600 per 100,000 population annually. The pattern of hand injuries bears some similarities and differences to published series. A comparison with data obtained in Hong Kong in the 1970s shows that major changes have occurred. We need continual surveillance of hand injury patterns.

Journal Article↗

PubMed1996

Effects of internal cleavages and mutations in the C-terminal region of NIa protease of turnip mosaic potyvirus on the catalytic activity.

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease processing the viral polyprotein into functional proteins. It has been shown that the NIa C-terminal 27-kDa protease cleaves itself between Ser-223 and Gly-224 to generate a 25-kDa protein lacking the C-terminal 20 amino acids. We have found a second internal cleavage near the C-terminus resulting in the degradation of the 25-kDa protein into a 24-kDa protein. Substitution of the active site Asp-81 or Cys-151 with Asn or Ser, respectively, prevented the second cleavage, suggesting that the internal cleavage is also due to the proteolytic activity of the NIa protease. This second internal cleavage was found to occur between Thr-207 and Ser-208, eliminating the C-terminal 36 amino acids from the 27-kDa protease. The proteolytic activity of the 24-kDa protein was not detected at all when it was measured using a nonapeptide containing the cleavage site between 6K1 and Cl as a substrate, suggesting that the C-terminal region between residues 208 and 223 contains essential amino acids for the processing of 6K1-Cl polyprotein. The deletion analyses of the C-terminal region revealed that at least 217 amino acids from the N-terminus are required for the catalytic activity of the NIa protease. The point mutation of Trp-212 to Ser, Gly-213 to Ser, or Ile-217 to Asp drastically abolished the catalytic activity, demonstrating that Trp-212, Gly-213, and Ile-217 are important for the processing of 6K1-Cl polyprotein.

Amino Acid Sequence↗

PubMed1996

Characterization of Nla protease from turnip mosaic potyvirus exhibiting a low-temperature optimum catalytic activity.

Nuclear inclusion protein a (Nla) protease of turnip mosaic potyvirus is responsible for the processing of the viral polyprotein into functional proteins. The Nla protease was found to exhibit its optimum catalytic activity at approximately 15 degrees and a bell-shaped pH-dependent activity profile with a maximum at approximately pH 8.5. Kinetic studies showed that both Km and V(max) values were lower at 12 than at 25 degrees in all three different pH conditions, pH 7.0, 7.4, and 8.3, indicating that the higher activity at 12 degrees is due to the lower value of Km. Interestingly, the self-cleavage of the 27-kDa protease to generate the 25-kDa protease occurred more rapidly at 25 than at 12 degrees, implying that the C-terminal self-cleavage site may interfere with the binding of the peptide substrate to the active site of the protease. Mutations and deletions at the C-terminal cleavage site had no effect on the temperature dependence of the proteolytic activity, demonstrating that the C-terminal self-cleavage is not related to the low-temperature optimum catalytic activity. The fluorescence measurement of the Nla protease upon temperature variation revealed that the protease undergoes a large conformational change between 2 and 42 degrees and a drastic transition near 45 degrees, suggesting that the low-temperature optimum catalytic activity is due to the highly flexible structure of the Nla protease.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary x-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B.

The delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 36.48 angstrom, b = 74.30 angstrom, c = 96.02 angstrom, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Angstrom resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.

Crystallography, X-Ray↗

PubMed1996

The SH3-domain protein Bem1 coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae.

The mating mitogen-activated protein kinase (MAPK) cascade has three major outputs prior to fusion: transcriptional activation of many genes, cell cycle arrest in the G1 phase, and polarized growth. Bem1 localizes near the cortical actin cytoskeleton and is essential for polarized growth during mating. Here we show that Bem1 is required for efficient signal transduction and coordinates MAPK cascade activation with G1 arrest and mating. bem1delta null mutants are defective in G1 arrest and transcriptional activation in response to mating pheromone. Bem1 protein stimulates Fus3 (MAPK) activity and associates with Ste5, the tethering protein essential for activation of the MAPK kinase kinase Ste11. Bem1-Ste5 complexes also contain Ste11, Ste7 (MAPK kinase), and Fus3, suggesting that Ste5 localizes the MAPK cascade to Bem1. Strikingly, Bem1 also copurifies with Far1, a Fus3 substrate required for G1 arrest and proper polarized growth during mating. These and other results suggest that Bem1 may cross-link the Ste5-MAPK cascade complex to upstream activators and specific downstream substrates at the shmoo tip, thus enabling efficient circuitry for G1 arrest and mating.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Cloning, sequencing and expression of the gene encoding a major allotypic preprocarboxypeptidase A from bovine pancreas.

A cDNA encoding one of two major allotypic forms of bovine pancreatic preprocarboxypeptidase A (preproCPA) has been cloned, and its entire nucleotide (nt) sequence determined. The cloned cDNA contains a 26-nt 5'-noncoding region, a 1260-nt open reading frame and a 51-nt 3'-noncoding region. The amino acid (aa) sequence deduced from the gene sequence contains Ile179, Ala228 and Val305 at allotypic aa residues. The procarboxypeptidase A (proCPA) was produced in yeast and secreted into the medium. Upon treatment with trypsin, proCPA generated an active enzyme with the same size as the mature carboxypeptidase A (CPA), as analyzed by use of anti-CPA polyclonal antibody.

Amino Acid Sequence↗

PubMed1995

Expression, purification, and identification of a novel self-cleavage site of the Nla C-terminal 27-kDa protease of turnip mosaic potyvirus C5.

The gene encoding the C-terminal protease domain (27 kDa) of the nuclear inclusion protein a of turnip mosaic potyvirus C5 was cloned and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. Two forms of the protease (27 and 25 kDa) were purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography and exhibited the specific proteolytic activity when a synthetic undecapeptide, Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-Asn-Glu, or an in vitro translation product of the polyprotein containing the cleavage site between the nuclear inclusion protein b and the capsid protein, was used as a substrate. The purified proteases showed a Km of 1.15 +/- 0.16 mM and a Vmax of 0.74 +/- 0.091 mumol/mg/min with the synthetic peptide substrate. The 25-kDa protein was found to be generated by the cleavage between Ser223 and Gly224 near the C-terminus of the 27-kDa protease and to retain the specific proteolytic activity. The point mutation of Asp81 or Cys151, two putative active site residues in the 27-kDa protease, to Asn or Ser, respectively, prevented the generation of the 25-kDa protein and diminished the proteolytic activity of the protease drastically, suggesting that the 27-kDa protease cleaves itself between Ser223 and Gly224 to generate the 25-kDa protein.

Amino Acid Sequence↗

PubMed1995

Transactivation ability of p53 transcriptional activation domain is directly related to the binding affinity to TATA-binding protein.

Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Gal4 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD-mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Gal4-p53 TAD also impair the binding activity to TBP severely. Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation deficient mutant type.

Amino Acid Sequence↗

PubMed1995

Mechanism of corepressor-mediated specific DNA binding by the purine repressor.

The modulation of the affinity of DNA-binding proteins by small molecule effectors for cognate DNA sites is common to both prokaryotes and eukaryotes. However, the mechanisms by which effector binding to one domain affects DNA binding by a distal domain are poorly understood structurally. In initial studies to provide insight into the mechanism of effector-modulated DNA binding of the lactose repressor family, we determined the crystal structure of the purine repressor bound to a corepressor and purF operator. To extend our understanding, we have determined the structure of the corepressor-free corepressor-binding domain of the purine repressor at 2.2 A resolution. In the unliganded state, structural changes in the corepressor-binding pocket cause each subunit to rotate open by as much as 23 degrees, the consequences of which are the disengagement of the minor groove-binding hinge helices and repressor-DNA dissociation.

Allosteric Regulation↗

PubMed1995

Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

In order to assess the roles of specific amino acid residues in the delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B during catalysis, we replaced aspartic acid 40 with asparagine (D40N) and tyrosine 16 with phenylalanine (Y16F) in the enzyme by site-directed mutagenesis. Both purified mutant enzymes resulted in profound decreases in catalytic activities, 10(3.3)-fold in the Y16F mutant and 10(6.2)-fold in the D40N mutant. Aspartic acid 40 and tyrosine 16 of the enzyme are the corresponding amino acids in the active site of the homologous enzyme from Comamonas testosteroni. Our results indicate that active-site residues of the two homologous enzymes are similar. This is opposite to the previous identification of a cysteine in an active site-directed photoinactivation study of the enzyme.

Amino Acid Sequence↗

PubMed1995

Percutaneous Kirschner-wire pinning for severely displaced distal radial fractures in children. A report of 157 cases.

Distal radial fractures are common in children. Recent outcome studies have cast doubt on the success of treatment by closed reduction and application of plaster. The most important risk factor for poor outcome is translation of the fracture. If a distal radial fracture is displaced by more than half the diameter of the bone at the fracture site it should be classified as high risk. We performed percutaneous Kirschner-wire pinning on 157 such high-risk distal radial fractures in children under 16 years of age. The predicted early and late failure rate was reduced from 60% to 14% and only 1.5% of patients had significant limitation of forearm movement of more than 15 degrees in the final assessment at a mean of 31 months after operation. There were no cases of early physeal closure or deep infection.

Adolescent↗

PubMed1994

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices.

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.

Amino Acid Sequence↗

PubMed1994

Mutagenesis of amino acid residues required for binding of corepressors to the purine repressor.

The corepressor-binding domain of the Escherichia coli purine repressor (PurR) is homologous with several periplasmic sugar-binding proteins. Four amino acids in PurR were investigated for a role in binding of corepressors. Three of the residues, Asp146, Arg196 and Asp275, are conserved in periplasmic binding proteins for ribose, glucose/galactose, and arabinose and function to bind sugars. A fourth amino acid, Trp147, required for corepressor binding to PurR, corresponds to residues in glucose/galactose, ribose, and arabinose that also have a role in sugar binding. The four mutations that were constructed perturbed the binding of both hypoxanthine and guanine thus providing evidence for a single corepressor site/PurR subunit. The decreased corepressor binding affinity resulted in reduced affinity of mutant repressors for operator DNA in vitro and decreased capacity for repression in vivo. The corepressor-binding site in PurR appears to be similar to the conserved ligand-binding sites in the three periplasmic sugar-binding proteins and in the LacI family of repressors.

Amino Acids↗

PubMed1994

Crystallization and preliminary X-ray analysis of an Escherichia coli purine repressor-hypoxanthine-DNA complex.

The purine repressor (PurR) is a DNA-binding protein, which together with a purine corepressor serves to regulate de novo purine and pyrimidine biosynthesis in Escherichia coli. PurR belongs to the structurally homologous lac repressor family of transcription regulators. A PurR-hypoxanthine-DNA complex has been crystallized, with DNA encompassing the high affinity purF operator site and which is 16 base-pairs long with 5'-deoxynucleoside overhangs on each complementary strand. The crystals diffract to better than 2.6 A and take the orthorhombic space group C222(1), with unit cell dimensions a = 175.9 A, b = 94.8 A and c = 81.8 A. The structure determination of this PurR-hypoxanthine-DNA complex will provide the first high resolution view of a Lacl member-DNA complex.

Bacterial Proteins↗

PubMed1994

Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae.

Ste5 is a Zn2+ finger-like protein thought to function before three kinases, Ste11 (a MEKK), Ste7 (a MEK), and Fus3 (a MAPK), in a conserved MAP kinase cascade required for mating in S. cerevisiae. Here, we present evidence that Ste5 forms a multikinase complex that joins these kinases for efficient Fus3 activation. By two-hybrid analysis, Ste11, Ste7, and Fus3 associate with different domains of Ste5, while Kss1, another MAPK, associates with the same domain as Fus3, thus implying that Ste5 simultaneously binds a MEKK, MEK, and MAPK. Ste5 copurifies with Ste11, Fus3, and a hypophosphorylated form of Ste7, and all four proteins cosediment in a glycerol gradient as if in a large complex. Ste5 also increases the amount of Ste11 complexed to Ste7 and Fus3 and is required for Ste11 to function. These results substantiate a novel signal transduction component that physically links multiple kinases within a single cascade.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.

Amino Acid Sequence↗

PubMed1994

Role of the purine repressor hinge sequence in repressor function.

A protease-hypersensitive hinge sequence in Escherichia coli purine repressor (PurR) connects an N-terminal DNA-binding domain with a contiguous corepressor-binding domain. Binding of one molecule of dimeric repressor to operator DNA protects the hinge against proteolytic cleavage. Mutations in the hinge region impair repressor function in vivo. Several nonfunctional hinge mutants were defective in low-affinity binding to operator DNA in the absence of corepressor as well as in high-affinity corepressor-dependent binding to operator DNA, although binding of corepressor was similar to binding of the wild-type repressor. These results establish a role for the hinge region in operator binding and lead to a proposal for two routes to form the holoPurR-operator complex.

Amino Acid Sequence↗