Effect of inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficiently inhibits a bacterial serine protease, subtilisin BPN'. We recently demonstrated that functional change in SSI was possible simply by replacing the amino acid residue at the reactive P1 site (methionine 73) of SSI. The present paper reports the additional effect of replacing methionine 70 at the P4 site of SSI (Lys73) on inhibitory activity toward two types of serine proteases, trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversion of methionine 70 at the P4 site of SSI(Lys73) to glycine or alanine resulted in increased inhibitory activity toward trypsin and lysyl endopeptidase, while replacement with phenylalanine weakened the inhibitory activity toward trypsin. This suggests that steric hindrance at the P4 site of SSI(Lys73) is an obstacle for its binding with trypsin. In contrast, the same P4 replacements had hardly any effect on inhibitory activity toward subtilisin BPN'. Thus the subsite structure of subtilisin BPN' is tolerant to these replacements. This contrast in the effect of P4 substitution might be due to the differences in the S4 subsite structures between the trypsin-like and the subtilisin-like proteases. These findings demonstrate the importance of considering structural complementarity, not only at the main reactive site but also at subsites of a protease, when designing stronger inhibitors.     

Prothrombinase assembly and S1 site occupation restore the catalytic activity of FXa impaired by mutation at the sodium-binding site

Two loop segments (183-189 and 221-225) in the protease domain of factor Xa contribute to the formation of a Na(+)-binding site. Studies with factor Xa indicate that binding of a single Na(+) ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr(225) with Pro diminishes sensitivity to Na(+). Using full-length factor Xa(Y225P), the allosteric relationship between the Na(+) site and other structural determinants in factor Xa and prothrombinase was investigated. Direct binding and kinetic measurements with probes that target the S1 specificity pocket indicate that assembly of the mutant in prothrombinase corrected the impaired binding of these probes observed with free factor Xa(Y225P). This appears to result from the apparent allosteric linkage between the factor Va, S1, and Na(+)-binding sites, since binding of the cofactor to membrane-bound factor Xa(Y225P) enhances binding at the S1 site and vice versa. Additional studies revealed that the internal salt bridge (Ile(16)-Asp(194)) of factor Xa(Y225P) is partially destabilized, a process that is reversible upon occupation of the S1 site. The data establish that alterations at the factor Xa Na(+)-binding site shift the zymogen-protease equilibrium to a more zymogen-like state, and as a consequence binding of S1-directed probes and factor Va are adversely affected. Therefore, the zymogen-like characteristics of factor Xa(Y225P) have allowed for the apparent allosteric linkage between the S1, factor Va, and Na(+) sites to become evident and has provided insight into the structural transitions which accompany the conversion of factor X to factor Xa.     

A point mutation at the ATP-binding site of the EGF-receptor abolishes signal transduction.

The EGF-receptor (EGF-R) is a transmembrane glycoprotein with intrinsic protein tyrosine kinase (TK) activity. To explore the importance of the receptor TK in the action of EGF, we have used transfected NIH-3T3 cells expressing either the normal human EGF-R or a receptor mutated at Lys721, a key residue in the presumed ATP-binding region. The wild-type receptor responds to EGF by causing inositol phosphate formation, Ca2+ influx, activation of Na+/H+ exchange and DNA synthesis. In contrast, the TK-deficient mutant receptor fails to evoke any of these responses. It is concluded that activation of the receptor TK is a crucial signal that initiates the multiple post-receptor effects of EGF leading to DNA synthesis. Furthermore, the results suggest that tyrosine phosphorylation plays a role in the activation of the phosphoinositide signalling system.     

Serpin B1 protects colonic epithelial cell via blockage of neutrophil elastase activity and its expression is enhanced in patients with ulcerative colitis

Serpin B1 is a monocyte neutrophil elastase (NE) inhibitor and is one of the most efficient inhibitors of NE. In the present study, we investigated the role of serpin B1 in the pathogenesis of ulcerative colitis by using clinical samples and an experimental model. The colonic expression of serpin B1 was determined by real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistological studies in both normal and inflamed mucosa from patients with ulcerative colitis. Serpin B1 mRNA expression was determined by real-time PCR in the mouse dextran sodium sulfate (DSS)-induced colitis model. Young adult mouse colonic epithelial (YAMC) cells were used to determine the role of serpin B1. Serpin B1 gene transfected YAMC cells were treated with H(2)O(2) to measure cell viability. The expression of NE was determined in YAMC cells treated with H(2)O(2). NE-silenced YAMC cells were also treated with H(2)O(2) and then measured for viability. Upregulated expression of serpin B1 in colonic mucosa was confirmed from patients with active ulcerative colitis. Immunohistochemical studies showed that serpin B1 expression was localized not only in inflammatory infiltration cells but also in epithelial cells. Serpin B1 mRNA expression was also increased in colonic mucosa of mouse DSS-induced colitis. Serpin B1-transfected YAMC cells were resistant against the treatment of H(2)O(2). H(2)O(2) treatment significantly induced NE in YAMC cells, and NE-silenced YAMC cells were also resistant against the treatment of H(2)O(2). These results suggest that serpin B1 may be a novel marker of active ulcerative colitis and may play an important role in the pathogenesis of inflammatory bowel disease.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.     

Redesign of Schistosoma mansoni NAD+ catabolizing enzyme: active site H103W mutation restores ADP-ribosyl cyclase activity

Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes that are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change, we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products (i.e., a value larger than observed for other members of this family such as CD38). Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provides new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge, however, in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR.     

alpha 1-Antitrypsin Christchurch, 363 Glu----Lys: mutation at the P'5 position does not affect inhibitory activity

alpha 1-Antitrypsin Christchurch was isolated from the plasma of a Cambodian woman who was heterozygous for this variant and for the normal M protein. Tryptic peptide maps revealed that the inhibitory-site peptide, 359-365 Ser-Ile-Pro-Pro-Glu,Val,Lys, was missing and replaced by two new peptides Ser-Ile-Pro-Pro,Lys and Val-Lys, indicating a mutation of 363 Glu----Lys. There was no obvious clinical condition associated with this new antitrypsin. Competition experiments showed that antitrypsin Christchurch reacted at the same rate as normal antitrypsin in the presence of limiting amounts of trypsin, chymotrypsin, thrombin and neutrophil elastase. Both inhibitors were inactivated by catalytic amounts of papain. This inactivation was due to cleavage at the phenylalanine residue at the P7 position, seven residues towards the N-terminal of the inhibitory site. A one-step ethanol extraction procedure is described for isolating the papain cleavage products.     

An abasic site analogue activates a c-Ha-ras gene by a point mutation at modified and adjacent positions.

Synthetic c-Ha-ras genes with an analogue of an abasic site in the first or the second position of codon 12, or in the second position of codon 61 were constructed and transfected into NIH3T3 cells. The genes with the lesions in codon 12 exhibited more focus formation than a normal c-Ha-ras gene, while the gene with the lesion in codon 61 did not. Transformed cells were isolated from the foci, and the c-Ha-ras genes present in the transformants were analysed. A point mutation to A in the modified position was found most frequently in the cases of ras genes modified in codon 12. Surprisingly, point mutations in the adjacent position were also detected. These results indicate that dTMP, and not dAMP, was mainly incorporated into the sites opposite to the abasic site analogue, and that incorrect deoxynucleotides were incorporated in the position adjacent to the abasic site analogue.     

Photochemical cross-linking of unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P site.

Acetylvalyl-, acetylphenylalanyl-, and formylmethionyl-tRNA which were derivatized at their 4-thiouridine residues with the photoaffinity label, p-azidophenacyl bromide, were nonenzymatically bound to salt-washed ribosomes. More than 90% of the binding was to the P site as judged by reactivity with puromycin. Subsequent irradiation (greater than 310 nm) of the tRNA-ribosome complexes resulted in the covalent linking of only the acetylvalyl-tRNA to the 30S subunit. Attachment was solely to the 16S RNA with an efficiency of cross-linking of 13--15%. Covalent linking was 90% inhibited by prior treatment with puromycin, showing that the covalent linking reaction had taken place at the P site. Cross-linking required irradiation and mRNA but was not dependent on the presence of the photoaffinity probe in the tRNA. tRNAs whose 4-thiouridine had been modified with unreactive analogues of p-azidophenacyl bromide or unmodified acetylvalyl-tRNA exhibited the same cross-linking behavior as photoaffinity probe-modified acetylvalyl-tRNA. Furthermore, even acetylvalyl-tRNA whose 4-thiouridine had been removed by treatment with H2O2 was quantitatively as active as unmodified tRNA. These results provide the first demonstration of direct photochemical cross-linking of tRNA to ribosomes.     

Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.     

Influence of charge distribution at the active site surface on the substrate specificity of human neutrophil protease 3 and elastase. A kinetic and molecular modeling analysis

The biological functions of human neutrophil protease 3 (Pr3) differ from those of neutrophil elastase despite their close structural and functional resemblance. Although both proteases are strongly cationic, their sequences differ mainly in the distribution of charged residues. We have used these differences in electrostatic surface potential in the vicinity of their active site to produce fluorescence resonance energy transfer (FRET) peptide substrates for investigating individual Pr3 subsites. The specificities of subsites S5 to S3' were investigated both kinetically and by molecular dynamic simulations. Subsites S2, S1', and S2' were the main definers of Pr3 specificity. Combinations of results for each subsite were used to deduce a consensus sequence that was complementary to the extended Pr3 active site and was not recognized by elastase. Similar sequences were identified in natural protein substrates such as NFkappaB and p21 that are specifically cleaved by Pr3. FRET peptides derived from these natural sequences were specifically hydrolyzed by Pr3 with specificity constants k(cat)/K(m) in the 10(6) m(-1) s(-1) range. The consensus Pr3 sequence may also be used to predict cleavage sites within putative protein targets like the proform of interleukin-18, or to develop specific Pr3 peptide-derived inhibitors, because none is available for further studies on the physiopathological function of this protease.     

beta-Secretase inhibitors: modification at the P4 position and improvement of inhibitory activity in cultured cells

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-570 and KMI-684 in which we replaced carboxylic acid groups at the P(1)(') position of KMI-420 and KMI-429, respectively, with tetrazole derivatives as carboxylic acid bioisosteres. These modifications improved significantly BACE1 inhibitory activity and chemical stability. In this study, the acidic tetrazole ring of the P(4) position of KMI-420 and KMI-570, respectively, was replaced with various hydrogen bond acceptor groups. We found BACE1 inhibitor KMI-574 that exhibited potent inhibitory activity in cultured cells as well as in vitro enzymatic assay.     

A point mutation at the subunit interface of hypoxanthine-guanine-xanthine phosphoribosyltransferase impairs activity: role of oligomerization in catalysis

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from Plasmodium falciparum catalyzes the phosphoribosylation of hypoxanthine, guanine and xanthine. The functionally active form of HGXPRT is a tetramer but interface residues do not contribute to catalysis. Here we report the characterization of an interface mutant Y96C, which has a decreased k(cat), an increase in the K(m) for phosphoribosyl pyrophosphate (PRPP) and no change in K(m) for the purine bases when compared to the wild type enzyme. The mutant enzyme does not tetramerize in the presence of PRPP, unlike the wild type in which the tetramer is stabilized by PRPP. This is the first report of a HGXPRT mutation, at a unique interface where non-adjacent subunits interact, that impairs catalysis.     

A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation

A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.     

A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.     

A point mutation in the ethylene-inducing xylanase elicitor inhibits the beta-1-4-endoxylanase activity but not the elicitation activity.

Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanase protein interacting directly with the plant cells. We cloned the gene encoding EIX protein and overexpressed it in insect cells. To determine the relationship between the two activities, substitution of amino acids in the xylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Gly) inhibited the beta-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis. However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained ability to induce ethylene biosynthesis and the hypersensitive response. Our data show that the xylanase activity of EIX elicitor can be separated from the elicitation process, as some of the mutants lack the former but retain the latter.     

HIV-1 protease inhibitors containing statine: inhibitory potency and antiviral activity.

Several series of chemically different inhibitors of the HIV-1 aspartyl protease have been described. Nevertheless despite the high in vitro potency showed, in most cases these inhibitors are unable to inhibit viral replication in infected cells. Penetration of the inhibitors across the cell membrane might account for their low antiviral activity. The relationship between inhibitory potency, antiviral activity and chemical structures of a series of oligopeptides containing statine or statine derivatives are presented here.     

Genetic defect in muscle phosphofructokinase deficiency. Abnormal splicing of the muscle phosphofructokinase gene due to a point mutation at the 5'-splice site

The genetic defect in muscle phosphofructokinase deficiency (type VII glycogenosis, Tarui disease) was investigated. Six cDNAs for muscle phosphofructokinase, including a full-length clone, were isolated from a non-amplified library of muscle from a patient. By sequence analysis of these clones, a 75-base in-frame deletion was identified. The rest of the sequence was identical to that of the normal cDNA, except for a silent base transition at position 516 (ACT (Thr) to ACC (Thr]. The deletion was located in the 3'-terminal region of exon 13 (numbered with reference to the rabbit muscle phosphofructokinase gene (Lee, C.-P., Kao, M.-C., French, B.A., Putney, S.D., and Chang, S.H. (1987) J. Biol. Chem. 262, 4195-4199]. Genomic DNA of the patient was amplified by polymerase chain reaction. Sequence analysis of the amplified DNA revealed a point mutation from G to T at the 5'-end of intron 13. This mutation changed the normal 5'-splice site of CAG:GTATGG to CAG:TTATGG. A cryptic splice site of ACT:GTGAGG located 75 bases upstream from the normal splice site was recognized and spliced in the patient.     

Alteration of apparent negative cooperativity of ATPase activity by alpha-subunit glutamine 173 mutation in yeast mitochondrial F1. Correlation with impaired nucleotide interaction at a regulatory site.

The first described alpha-subunit mutation of yeast mitochondrial F1 has been recently identified as a single Gln173----Leu substitution in a strongly conserved sequence (Falson, P., Maffey, L., Conrath, K., and Boutry, M. (1991) J. Biol. Chem. 266, 287-293). This mutation is shown here to greatly modify the biphasic pattern of ATPase activity as a function of pH: (i) the shoulder observed at acidic pH is significantly increased; (ii) the main peak, at alkaline pH, is markedly lowered; (iii) the optimal pH is shifted from 8.8 to 7.7. The mutation lowers both apparent negative cooperativity and sensitivity to azide inhibition which concomitantly increase when the assay pH decreases. Azide partial inhibition produces apparent negative cooperativity which can be further abolished by bicarbonate. The mutation increases both activation energies determined from biphasic Arrhenius plots. The mutation decreases the inactivation rate by 5'-p-fluorosulfonylbenzoyladenosine and abolishes the protection by nucleotide binding at the adenine-specific regulatory site. On the contrary, it does not modify the reactivity of 5'-p-fluorosulfonylbenzoylguanosine at the less-selective catalytic site. In addition, partial inactivation by 5'-p-fluorosulfonylbenzoyladenosine, as opposed to 5'-p-fluorosulfonylbenzoylguanosine, produces apparent negative cooperativity under conditions where unmodified-enzyme kinetics are noncooperative. The results show that alpha-Gln173 participates in nucleotide interaction at a regulatory site which controls the negative cooperativity of F1-ATPase activity.