Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein G columns were used to remove IgG from human plasma, and the effect on levels of factor XII, factor XI and prekallikrein was studied in functional tests. IgG was detected in PAGE immunoblot experiments with Fc-specific antibodies. Removal of the bulk of IgG in a procedure based on a low plasma dilution (1+2.5) allowed the passage of an IgG fraction along with the contact factors. This fraction was found to be present in higher amounts in plasma from patients with Crohn's disease (n=5) than in control plasma (n=12). In a previous study, PAGE immunoblot experiments showed that part of the prekallikrein was removed along with IgG when a higher plasma dilution (1+10.8) was used (Scand J Clin Lab Invest 1999; 59: 55-64). This observation was supported by results in the present work based on parallel assays with the peptide substrates S-2302 and Bz-Pro-Phe-Arg-pNA. The prekallikrein fraction removed was present in a functional state differing from the main part of prekallikrein by yielding kallikrein with a significantly increased activity against the substrate S-2366. This prekallikrein fraction was present in higher amounts in patient plasma than in control plasma. Part of the corresponding amidase activity was blocked by lima bean trypsin inhibitor, suggesting its presence in association with factor XI. The results also indicated that prekallikrein activator activity was connected with this fraction. With the high dilution procedure an extensive removal of IgG from the patient plasma was obtained compared to the control plasma.
...
PMID:Removal of IgG from normal plasma and plasma from untreated patients with active Crohn's disease--effect on levels of contact factors. 1088 96

In order to circumvent the difficulty encountered in the expression and purification of the recombinant products in E. coli system, we have developed a novel and facile method of removing the polyhistidine tag from target proteins after heterologous gene expression. The expression of a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) is taken as an exemplar to illustrate the basic rationales and protocols involved. In place of an enterokinase recognition site, a polyhistidine tag linked to an autocatalyzed site based on cleavage specificity of the serine protease flanking on the 5'-end of Tm-5 clone sequence was engineered before protein expression in E. coli system. Renaturation of the fusion protein after expression revealed that the recombinant protease had refolded successfully from the inclusion bodies. Upon autocleavage of the expressed protease, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed accordingly. The protein expressed and purified by this new strategy possesses a molecular weight of approximately 28,000 in accord with the expected value for this venom protease. Further characterization of the recombinant protein employing a variety of techniques which include immunoblot analysis, RP-HPLC, ESI-MS, and N-terminal amino acid sequencing all shows indistinguishable properties to those of the isolated native protease. Most noteworthy is that the recombinant Tm-5 protease also exhibits amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously.
...
PMID:Expression of a kallikrein-like protease from the snake venom: engineering of autocatalytic site in the fusion protein to facilitate protein refolding. 1097 23

Using site directed mutagenesis combined with chemical modification, we have developed a general and versatile method for the glycosylation of proteins which is virtually unlimited in the scope of proteins and glycans that may be conjugated and in which the site of glycosylation and the nature of the introduced glycan can be carefully controlled. We have demonstrated the applicability of this method through the synthesis of a library of 48 glycosylated forms of the serine protease subtilisin Bacillus lentus (SBL) as single, pure species. As part of our ongoing program to tailor the activity of SBL for use in peptide synthesis, we have screened these enzymes for activity against the esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl. Gratifyingly, 22 enzymes displayed greater than wild type (WT) activity. Glycosylation at positions 62, in the S2 pocket, resulted in five glycosylated forms of SBL that were 1.3- to 1.9-fold more active than WT. At position 217, in the S1' pocket, all glycosylations increased kcat/KM up to a remarkable 8.4-fold greater than WT for the glucosylated enzyme L217C-S-beta-Glc(Ac)3. Furthermore, the ratio of amidase to esterase activity, (kcat/KM)esterase/(kcat/KM)amidase (E/A), is increased relative to wild type for all 48 glycosylated forms of SBL. Again, the most dramatic changes are observed at positions 62 and 217 and L217C-S-beta-Glc(Ac)3 has an E/A that is 17.2-fold greater than WT. The tailored specificity and high activity of this glycoform can be rationalized by molecular modeling analysis, which suggests that the carbohydrate moiety occupies the S1' leaving group pocket and enhances the rate of deacylation of the acyl-enzyme intermediate. These glycosylated enzymes are ideal candidates for use as catalysts in peptide synthesis as they have greatly increased (kcat,KM)esterase and severely reduced (kcat/KM)amidase and will favor the formation of the amide bond over hydrolysis.
...
PMID:Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity. 1097 2

In an attempt to investigate the molecular basis of pyrazinamide hydrolysis by the PncA protein from Mycobacterium tuberculosis, we determined the pyrazinamidase activity of nine PncA mutants bearing a single amino acid substitution. Among them, three mutants (D8G, K96T and S104R) had virtually no activity (< or =0.004 unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06-0.25 unit/mg) and one (T167L) exhibited a wild-type activity (1.51 units/mg). The possible structural effects of these substitutions were assessed by analysing a three-dimensional model of the PncA protein constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp., an amidohydrolase which was found by hydrophobic cluster analysis to be closely related to PncA. In the PncA model, five of the mutated residues, Asp-8, Phe-13, Lys-96, Tyr-103 and Ser-104, were located within a 6 A sphere around the cysteine residue Cys-138, which could be the counterpart of the active cysteine residue Cys-177 found in the CSHase. Among the remaining mutated residues, Thr-61, Pro-69 and Ala-146 were found to be more distant from Cys-138 but were associated with structural elements contributing to the catalytic centre, whereas Thr-167 was situated in an alpha-helix located far from the putative active site. These data suggest that the decrease in pyrazinamidase activity observed in the PncA mutant proteins is well correlated with the structural modifications the mutations can cause in the environment of the putative active cysteine Cys-138.
...
PMID:Study of the structure-activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis. 1117 Oct 40

Two venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major crotalid snake species in Taiwan. The purified enzymes showed a strong beta-fibrinogenolytic activity, cleaving the beta-chain of fibrinogen molecules specifically. They also showed strong kallikrein-like activity in vitro, releasing bradykinin from kininogen. The purified enzymes did not coagulate human plasma, yet decreasing fibrinogen levels in plasma and prolonging bleeding without formation of fibrin clots, indicating that both proteases have specificities different from thrombin and the thrombin-like proteases of snake venom reported previously. They also exhibit amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and found to be more stable when compared with ancrod and thrombin. Intravenous injection of either protease was shown to lower blood pressure in experimental rats. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases.
...
PMID:Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities. 1123 64

Concomitant activity improvement of an evolved enzyme toward two very different ester substrates was achieved when a unique combination of functional periplasmic enzyme expression in Escherichia coli, random mutagenesis, DNA shuffling and cell-based kinetic screenings was applied. Specifically, we focused on the conversion of subtilisin E into an enzyme with broader esterase activity as opposed to its native amidase activity. Cell-based microtiter assays were performed on N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as on the tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide. After a single modified cycle of directed molecular evolution, we isolated a number of clones exhibiting increased activity toward Phe-NPE. In the following rounds of screenings, mutants with improved activity on Phe-NPE were also tested on S1'A. Three mutants were identified with increased esterolytic activity on Phe-NPE and S1'A, while having similar amidase activity to that of the parental enzymes. Because the two ester substrates are structurally distinct, we have evolved a more general esterolytic subtilisin and this may have important applications in synthesis.
...
PMID:Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression. 1174 13

We developed a protocol for efficient expression of the functional serine protease, subtilisin E, in Escherichia coli periplasm that permits direct in vivo measurement of the enzyme's catalytic activity. Activity assays and SDS-PAGE/Western blot analysis showed that the levels of expressed subtilisin varied and were correlated with both the culture conditions and the induction procedures. The highest level of subtilisin expression was achieved at 0.10-0.15% (w/v) of arabinose as inducer and a temperature of 20-22 degrees C, and was ca. eightfold higher as compared to the expression level at 30 degrees C. Cultivation of bacterial cells to a steady state of balanced growth before induction was required for uniform subtilisin expression in cell cultures growing in wells of microtiter plates. Amidase and esterase cell-based kinetic assays on microtiter plates were developed based on the direct measurement of subtilisin activity in vivo. Intact E. coli cells displaying wild-type, dimethylformamide-resistant, and temperature-resistant subtilisins were assayed on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-acetyl-Phe-p-nitrophenyl ester for their amidase and esterase activity, respectively. Additionally, the periplasmic fractions were isolated from the three E. coli strains expressing the respective subtilisins and tested for amidase activity. The amidase activity of the three subtilisins was ca. 15-fold higher than the esterolytic activity when measured in both the intact cells and in the periplasmic fractions. The strategy combining periplasmic expression of subtilisins with two cell-based kinetic assays permits rapid screening of subtilisin mutant libraries for desired activities.
...
PMID:A strategy for in vivo screening of subtilisin E reaction specificity in E. coli periplasm. 1200 Nov 68

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.
...
PMID:Part of purified human plasma kallikrein removed together with a remaining IgG fraction--immunoblot experiments and functional tests. 1200 9

The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene ( pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM. The natural function of Pam remains unclear. Chymostatin ( K(i)<0.3 microM) and Pefabloc SC ( K(i) not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.
...
PMID:Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia. 1202 98

A flexible loop (residues 328-339), presumably covering the active site upon substrate binding, has been revealed in 3,4-dihydroxyphenylalanine decarboxylase by means of kinetic and structural studies. The function of tyrosine 332 has been investigated by substituting it with phenylalanine. Y332F displays coenzyme content and spectroscopic features identical to those of the wild type. Unlike wild type, during reactions with l-aromatic amino acids under both aerobic and anaerobic conditions, Y332F does not catalyze the formation of aromatic amines. However, analysis of the products shows that in aerobiosis, l-aromatic amino acids are converted into the corresponding aromatic aldehydes, ammonia, and CO(2) with concomitant O(2) consumption. Therefore, substitution of Tyr-332 with phenylalanine results in the suppression of the original activity and in the generation of a decarboxylation-dependent oxidative deaminase activity. In anaerobiosis, Y332F catalyzes exclusively a decarboxylation-dependent transamination of l-aromatic amino acids. A role of Tyr-332 in the Calpha protonation step that catalyzes the formation of physiological products has been proposed. Furthermore, Y332F catalyzes oxidative deamination of aromatic amines and half-transamination of d-aromatic amino acids with k(cat) values comparable with those of the wild type. However, for all the mutant-catalyzed reactions, an increase in K(m) values is observed, suggesting that Y --> F replacement also affects substrate binding.
...
PMID:Mutation of tyrosine 332 to phenylalanine converts dopa decarboxylase into a decarboxylation-dependent oxidative deaminase. 1211 7


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---