In vivo formation and stability of engineered disulfide bonds in subtilisin

Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.     

Mutagenesis at the ad-3A and ad-3B loci haploid UV-sensitive strains of Neurospora crassa. III. Comparison of dose-response curves for inactivation and mutation induced by gamma-rays

Gamma-Ray-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 6 different UV-sensitive strains and a standard wild-type strain. The 6 strains show varying degrees of sensitivity to gamma-ray-induced inactivation, with the relative sensitivity at 37% survival being uvs-6 greater than upr-1 greater than uvs-2 greater than uvs-3 greater than wild-type greater than uvs-5 greater than uvs-4. Studies on the induction of ad-3 mutants by gamma-rays show that when the dose-response curve (expressed in terms of ad-3 mutants among the surviving colonies) of the UV-sensitive strains are compared with wild-type, the 2 excision-repair-deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, uvs-3 exhibits reduced ad-3 mutant frequencies whereas both uvs-4 and uvs-5 show lower mutant frequencies than wild-type.     

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography

Recombinant wild-type protease of human immunodeficiency virus, type [(HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild-type protease. A third mutant protease (Arg-87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.     

Insecticidal activity and processing in larval gut juices of genetically engineered 130-kDa proteins of Bacillus thuringiensis subsp. aizawai.

The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S. litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S. litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.     

Purification and polymerization properties of two lethal yeast actin mutants

The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth. Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere [Buzan, J., Du, J., Karpova, T., and Frieden, C. (1999) Proc. Natl. Acad. Sci. USA 96, 2823-2827]. The mutations, in muscle actin numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament. The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth. The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca. 4 microM) without a large change in the ability to form nuclei for the nucleation-elongation process.     

Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.     

Effects of mutations in poliovirus 3Dpol on RNA polymerase activity and on polyprotein cleavage.

A series of short insertion mutations was introduced into the poliovirus gene for 3Dpol at a number of different locations. When substituted for wild-type sequences in a full-length, infectious cDNA and tested for infectivity, all 3D mutants were nonviable. The mutant cDNAs were introduced into a bacterial plasmid designed to direct the expression of poliovirus 3CD, a viral protein composed of contiguous protease and RNA polymerase sequences. Bacteria transformed with these plasmids all expressed similar amounts of 3CD, and all mutant proteins cleaved themselves to generate wild-type 3Cpro and mutant 3Dpol polypeptides with approximately the same efficiency as wild-type 3CD. The released mutant 3Dpol proteins were all defective in RNA-dependent RNA polymerase activity in vitro. Uncleaved 3CD is a protease required for processing the viral capsid protein precursor, P1. In an in vitro assay of P1 cleavage activity, some of the mutant 3CD proteins expressed in Escherichia coli showed normal activity, while others were clearly inactive. Thus, alterations in the sequence and/or folding of different regions of the 3D protein have differential effects on its various activities.     

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).     

The ++Sinorhizobium meliloti lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.     

Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions.

Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp. strain B18'. The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664). The amino acid sequence of this protease was compared with those of other serine proteases. A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed. An even more thermostable mutant enzyme was created by introducing a point mutation. When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%). The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition. These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.     

Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain.

Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure. The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine. The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity. Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme. However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values. However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes. Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme. Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme. Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value. The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.     

Non-Mendelian inheritance induced by gene amplification in the germ nucleus of Paramecium tetraurelia

A genetic investigation of strain d4-95, which carries a recessive mutant allele (pwB(95)) of pawn-B, one of the controlling elements of voltage-dependent calcium channels in Paramecium tetraurelia, revealed a non-Mendelian feature. Progeny of the cross between d4-95 and wild type often expressed a clonally stable mutant phenotype, even when they had a wild-type gene. The mutant phenotype was also expressed after self-fertilization of theoretical wild-type homozygotes recovered from the cross. Our molecular analysis demonstrated that the copy number of the mutant pwB gene in the micro- and macronucleus of d4-95 was much greater than that of the wild type. Most of the amplified, extra pwB gene copies in d4-95 were heritable independently from the original pwB locus. Repeated backcrossing of d4-95 with the wild type to dilute extra pwB genes in the strain produced segregants with a completely normal Mendelian trait in testcrosses. These results strongly suggest that a non-Mendelian inheritance of d4-95 was induced by gene amplification in the micronucleus.     

Influence of the plant defense response to Escherichia coli O157:H7 cell surface structures on survival of that enteric pathogen on plant surfaces

Consumption of fresh and fresh-cut fruits and vegetables contaminated with Escherichia coli O157:H7 has resulted in hundreds of cases of illness and, in some instances, death. In this study, the influence of cell surface structures of E. coli O157:H7, such as flagella, curli fimbriae, lipopolysaccharides, or exopolysaccharides, on plant defense responses and on survival or colonization on the plant was investigated. The population of the E. coli O157:H7 ATCC 43895 wild-type strain was significantly lower on wild-type Arabidopsis plants than that of the 43895 flagellum-deficient mutant. The population of the E. coli O157:H7 43895 flagellum mutant was greater on both wild-type and npr1-1 mutant (nonexpressor of pathogenesis-related [PR] genes) plants and resulted in less PR gene induction, estimated based on a weak β-glucuronidase (GUS) signal, than did the 43895 wild-type strain. These results suggest that the flagella, among the other pathogen-associated molecular patterns (PAMPs), made a substantial contribution to the induction of plant defense response and contributed to the decreased numbers of the E. coli O157:H7 ATCC 43895 wild-type strain on the wild-type Arabidopsis plant. A curli-deficient E. coli O157:H7 86-24 strain survived better on wild-type Arabidopsis plants than the curli-producing wild-type 86-24 strain did. The curli-deficient E. coli O157:H7 86-24 strain exhibited a GUS signal at a level substantially lower than that of the curli-producing wild-type strain. Curli were recognized by plant defense systems, consequently affecting bacterial survival. The cell surface structures of E. coli O157:H7 have a significant impact on the induction of differential plant defense responses, thereby impacting persistence or survival of the pathogen on plants.     

Arginine 177 is involved in Mn(II) binding by manganese peroxidase.

Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were approximately 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H(2)O(2) and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were approximately 10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were approximately 22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.     

An active-site mutation (Gly633-->Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum.

Dipeptidyl peptidase IV (DPPIV), a serine protease expressed on the cell surface, is deficient in a Fischer rat substrain. Northern blot analysis showed no difference in the size and amount of DPPIV mRNA between normal (344/NC) and deficient (344/CRJ) rats. Cloning and sequencing of DPPIV cDNAs revealed a G to A transition at nucleotide 1897 in the cDNA sequence of 344/CRJ, which leads to substitution of Gly633-->Arg in the active-site sequence Gly629-Trp-Ser-Tyr-Gly633 determined for the wild-type DPPIV [Ogata, S., Misumi, Y., Takami, N., Oda, K., & Ikehara, Y. (1992) Biochemistry 31, 2582-2587]. Pulse-chase experiments with hepatocytes showed that the wild-type DPPIV was initially synthesized as a 103-kDa form with high-mannose-type oligosaccharides, which was processed to a mature form of 109 kDa with the complex type during intracellular transport. In contrast, the mutant DPPIV, although being synthesized as the 103-kDa form, was rapidly degraded without being processed to the mature form. Site-directed mutagenesis of the wild-type and mutant cDNAs and their transfection/expression in COS-1 cells confirmed that the single substitution of Gly633-->Arg is sufficient to cause the rapid intracellular degradation of DPPIV. Immunoelectron-microscopic observations showed that the mutant DPPIV was detectable only in the endoplasmic reticulum (ER), in contrast to the distribution of the wild-type DPPIV in the Golgi complex and on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the alpha subunit

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg much less than Lys much less than Gln much less than Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase alpha subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2(+)-independent kinase activity (5.46 +/- 0.88%) compared to the wild-type Arg283 (1.86 +/- 0.71%) and the Gln283 mutant (2.15 +/- 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2(+)-independent activity, the Arg283 kinase attained maximal Ca2(+)-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2(+)-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2(+)-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

Synthesis, purification, and characterization of an Arg152----Glu site-directed mutant of recombinant human blood clotting factor VII

Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg152-Ile153. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, we have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg152----Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. Purified mutant factor VII was indistinguishable from plasma-derived or recombinant wild-type factor VII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated as a single band with an apparent molecular weight of 50,000. The average specific activity of several mutant factor VII preparations was 0.00025 unit/micrograms, or 0.01% of that observed for recombinant wild-type factor VII preparations. The clotting activity of mutant factor VII was, however, completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease.(ABSTRACT TRUNCATED AT 250 WORDS)