scholarly journals Roles of Amino Acids 161 to 179 in the PSE-4 Ω Loop in Substrate Specificity and in Resistance to Ceftazidime

1998 ◽  
Vol 42 (10) ◽  
pp. 2576-2583 ◽  
Author(s):  
Christian Therrien ◽  
Francois Sanschagrin ◽  
Timothy Palzkill ◽  
Roger C. Levesque
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
High Activity ◽  
Amino Acid Substitutions ◽  
Hydrolyzing Enzymes ◽  
Hydrolysis Of

ABSTRACT The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Ω loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Ω loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.

scholarly journals Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

2010 ◽  
Vol 55 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Akiko Shimizu-Ibuka ◽  
Mika Oishi ◽  
Shoko Yamada ◽  
Yoshikazu Ishii ◽  
Kiyoshi Mura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Binding Site ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Hydrolysis Of

ABSTRACTToho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced inEscherichia colistrains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

scholarly journals The CaaX Proteases, Afc1p and Rce1p, Have Overlapping but Distinct Substrate Specificities

2000 ◽  
Vol 20 (12) ◽  
pp. 4381-4392 ◽  
Author(s):  
Cynthia Evans Trueblood ◽  
Victor L. Boyartchuk ◽  
Elizabeth A. Picologlou ◽  
David Rozema ◽  
C. Dale Poulter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Single Amino Acid ◽  
In Vitro Assays ◽  
Sequence Motif ◽  
Amino Acid Substitutions ◽  
In The Wild

ABSTRACT Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a1, the a2, or the X position of the a-factor Ca1a2X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a1 position, V, L, I, C, or M at the a2 position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a1 substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

scholarly journals S-Acylation of Proteins of Coronavirus and Influenza Virus: Conservation of Acylation Sites in Animal Viruses and DHHC Acyltransferases in Their Animal Reservoirs

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 669
Author(s):  
Dina A. Abdulrahman ◽  
Xiaorong Meng ◽  
Michael Veit
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Ion Channels ◽  
Substrate Specificity ◽  
Influenza A ◽  
3D Structure ◽  
Viral Fitness ◽  
Amino Acid Substitutions ◽  
Essential Function ◽  
Animal Reservoirs

Recent pandemics of zoonotic origin were caused by members of coronavirus (CoV) and influenza A (Flu A) viruses. Their glycoproteins (S in CoV, HA in Flu A) and ion channels (E in CoV, M2 in Flu A) are S-acylated. We show that viruses of all genera and from all hosts contain clusters of acylated cysteines in HA, S and E, consistent with the essential function of the modification. In contrast, some Flu viruses lost the acylated cysteine in M2 during evolution, suggesting that it does not affect viral fitness. Members of the DHHC family catalyze palmitoylation. Twenty-three DHHCs exist in humans, but the number varies between vertebrates. SARS-CoV-2 and Flu A proteins are acylated by an overlapping set of DHHCs in human cells. We show that these DHHC genes also exist in other virus hosts. Localization of amino acid substitutions in the 3D structure of DHHCs provided no evidence that their activity or substrate specificity is disturbed. We speculate that newly emerged CoVs or Flu viruses also depend on S-acylation for replication and will use the human DHHCs for that purpose. This feature makes these DHHCs attractive targets for pan-antiviral drugs.

scholarly journals Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480 ◽  
Author(s):  
E Moran ◽  
B Zerler ◽  
T M Harrison ◽  
M B Mathews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Point Mutations ◽  
Apparent Molecular Weight ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Transformation Function ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

PREDICTION OF PATHOGENIC EFFECT FOR AMINO ACID SUBSTITUTIONS IN BRCA1 AND BRCA2 PROTEINS USING MNA DESCRIPTORS AND NAIVE BAYES CLASSIFIER

2020 ◽  
pp. 250-252
Author(s):  
D. Filimonov ◽  
A. Lagunin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Sequence ◽  
Amino Acid Substitutions ◽  
Classification Models ◽  
Bayes Classifier ◽  
Brca1 And Brca2 ◽  
Chemical Structures ◽  
Pathogenic Effect ◽  
Pathogenic Effects

It is advisable to use data peptide's chemical structures with amino acids (AMA) substitution and the corresponding sections of the protein sequence without mutation to construct classification models predicting the pathogenic effects AMA substitutions based on MNA descriptors.

scholarly journals Generation of Recombinant Rotavirus with an Antigenic Mosaic of Cross-Reactive Neutralization Epitopes on VP4

2008 ◽  
Vol 82 (13) ◽  
pp. 6753-6757 ◽  
Author(s):  
Satoshi Komoto ◽  
Masanori Kugita ◽  
Jun Sasaki ◽  
Koki Taniguchi
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Reverse Genetics ◽  
Amino Acid Substitutions ◽  
Neutralization Epitope ◽  
First Report ◽  
P Type

ABSTRACT Recombinant rotavirus (RV) with cDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VP5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.

scholarly journals THE AMINO ACID COMPOSITION OF HYPERTENSIN II AND ITS BIOCHEMICAL RELATIONSHIP TO HYPERTENSIN I

1956 ◽  
Vol 104 (2) ◽  
pp. 183-191 ◽  
Author(s):  
Kenneth E. Lentz ◽  
Leonard T. Skeggs ◽  
Kenneth R. Woods ◽  
Joseph R. Kahn ◽  
Norman P. Shumway
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Composition ◽  
Acid Content ◽  
Amino Acid Content ◽  
Converting Enzyme ◽  
Terminal Sequence ◽  
Carboxyl Terminal ◽  
Hydrolysis Of

Preparations of hypertensin II, obtained from the treatment of hypertensin I by the action of the hypertensin converting enzyme of plasma and purified by countercurrent distribution, were quantitatively analyzed for their amino acid content. Chromatography on ion exchange columns showed the presence of equimolar amounts of aspartic acid, proline, valine, isoleucine, tyrosine, phenylalanine, histidine, and arginine. Hypertensin I was found to contain one mole of leucine and one mole of histidine in addition to the amino acids of hypertensin II. These two amino acids were isolated from the conversion products of hypertensin I and identified as the peptide histidylleucine. Carboxypeptidase digestion of hypertensin I showed the carboxyl terminal sequence of amino acids to be residue-phenylalanyl-histidylleucine. Similar studies of hypertensin II demonstrated residue-phenylalanine. It was concluded that the conversion of hypertensin I by the plasma hypertensin converting enzyme involved hydrolysis of the phenylalanyl-histidine bond to form hypertensin II and histidylleucine. The further removal by carboxypeptidase of phenylalanine from hypertensin II destroyed all of the vasoconstrictor activity.

scholarly journals The isolation of a product of hydrolysis of the proteins hitherto undescribed

1925 ◽  
Vol 98 (687) ◽  
pp. 58-65 ◽  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Free Amino Acid ◽  
Convenient Method ◽  
Free Amino ◽  
Barium Carbonate ◽  
Present Communication ◽  
Hydrolysis Products ◽  
Hydrolysis Of

Some time ago, two of the authors of the present communication, in seeking a method for the separation of the amino-acids from the carbohydrates, found that under certain conditions the former could be readily separated in the form of the barium salts of their carbamates, a class of compounds originally described by Siegfried. As these carbamates, on heating with water, are readily decomposed into barium carbonate and the free amino-acid, it was suggested that a convenient method might be evolved, using the formation of these compounds as a basis, for the separation of the hydrolysis products of the proteins.* This suggestion was followed up, and a method was subsequently elaborated and applied to the separation of the hydrolysis products of gelatin by one of the authors in conjunction with Miss H. L. Kingston. Since the publication of the two papers just quoted, the researches on the use of the “carbamate method,” as it may be conveniently called, have been continued, and promise results, which may ultimately lead to a satisfactory separation of most of the hydrolysis products of the proteins when only relatively small amounts of material are available for investigation. During the course of this work the base, which is the chief subject discussed in this paper, was discovered.

Substrate Preferences for Antiplasmin Cleaving Enzyme Hydrolysis of Peptides Derived from the α2-Antiplasmin Sequence.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1704-1704
Author(s):  
Kenneth W. Jackson ◽  
Victoria J. Christiansen ◽  
Kyung N. Lee ◽  
Christina F. Mason ◽  
Patrick A. McKee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Enzyme Hydrolysis ◽  
Peptide Sequence ◽  
Peptide Hydrolysis ◽  
Cleavage Rate ◽  
Amino Terminal ◽  
Substrate Preferences ◽  
Polymorphic Position ◽  
Hydrolysis Of

Abstract Antiplasmin cleaving enzyme (APCE) is a proteinase that specifically, but slowly cleaves the Pro-Asn bond in long-form α2-antiplasmin (Met-α2AP) in human plasma. This slow cleavage produces a steady-state plasma mixture of Met-α2AP and an N-terminally shortened form of antiplasmin, Asn-α2AP. The Asn-α2AP form crosslinks to fibrin ~13-fold faster than Met-α2AP. A faster crosslink rate causes a greater number of antiplasmin molecules to become bound during fibrin formation, thereby enhancing resistance to fibrinolysis. Inhibition of plasma APCE may decrease the number of antiplasmin molecules crosslinked and result in clots that are more easily removed during fibrinolysis. Therefore, an inhibitor specific for APCE could potentially be used to regulate fibrinolysis. Human Met-α2AP exists in two polymorphic forms at position six in the mature sequence, with arginine predominant, and tryptophan accounting for a lesser percentage. We have determined the relative cleavage rates of synthetic peptides from a peptide library that span the cleavage site. The peptides contained all common amino acids except cysteine in the polymorphic position (P7 position). Arg was the optimal amino acid in this position with a relative cleavage rate ~5–10-fold faster than other amino acids except Lys, which was ~70% of the Arg rate. The P7 position Arg enhancement was also observed when Arg was in the P6 or P5 position, but no enhancement was observed when Arg was moved to positions P8, P4, P3 or P2. It was also determined that APCE is preferentially an endoproteinase rather than an aminodipeptidase, with a 10-fold greater rate of hydrolysis of the internal Pro-Asn bond in the Met-α2AP 1–17 peptide sequence MEPLGRQLTSGP-NQEQV over the Pro-Asn bond penultimate to the amino-terminal bond in the Met-α2AP 11–27 peptide sequence GP-NQEQVSPLTLLKLGN in peptide hydrolysis experiments. We conclude that APCE inhibitors designed with a positive charge placed upstream of the Pro-X scissile bond equivalent to five to seven amino acid residues may prove to be highly potent and specific. In addition, such inhibitors should also prove useful for blocking the activity of the closely related enzyme fibroblast activation protein. This work was supported by NIH grant HL072995.

scholarly journals Chlorella Virus-Encoded Deoxyuridine Triphosphatases Exhibit Different Temperature Optima

2005 ◽  
Vol 79 (15) ◽  
pp. 9945-9953 ◽  
Author(s):  
Yuanzheng Zhang ◽  
Hideaki Moriyama ◽  
Kohei Homma ◽  
James L. Van Etten
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Divalent Cations ◽  
Catalytic Efficiency ◽  
Amino Acid Substitutions ◽  
Temperature Optimum ◽  
Docking Simulations ◽  
Chlorella Virus ◽  
Temperature Optima

ABSTRACT A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg2+ for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a Km of 11.7 μM, a turnover k cat of 6.8 s−1, and a catalytic efficiency of k cat/Km = 5.8 × 105 M−1 s−1. dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37°C) than PBCV-1 dUTPase (50°C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81→Ser81 and Thr84→Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84→Arg84, Glu81→Ser81, and Glu81→Ser81 plus Thr84→Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55°C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.

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