scholarly journals Change in Substrate Preference of Streptomyces Aminopeptidase through Modification of the Environment around the Substrate Binding Site

2006 ◽  
Vol 72 (12) ◽  
pp. 7962-7967 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Binding Site ◽  
Substrate Binding ◽  
The Other ◽  
Substrate Preference ◽  
Wild Type ◽  
Substrate Binding Site ◽  
Hydrolysis Of

ABSTRACT We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k cat values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively).

scholarly journals Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity

2004 ◽  
Vol 382 (1) ◽  
pp. 191-198 ◽  
Author(s):  
Juliana Martha SÁ ◽  
Lucimara CHIOATO ◽  
Tatiana Lopes FERREIRA ◽  
Arthur H. C. de OLIVEIRA ◽  
Roberto RULLER ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Binding Site ◽  
Inclusion Bodies ◽  
Substrate Binding ◽  
Membrane Damage ◽  
Cd Spectra ◽  
Wild Type ◽  
Mutant Proteins ◽  
Substrate Binding Site ◽  
Independent Mechanism

BthTx-I (bothropstoxin-I) is a myotoxic Lys49-PLA2 (phospholipase A2 with Lys49) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca2+-independent mechanism. The highly conserved Phe5/Ala102/Phe106 motif in the hydrophobic substrate-binding site of the Asp49-PLA2s is substituted by Leu5/Val102/Leu106 in the Lys49-PLA2s. The Leu5/Val102/Leu106 triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe5/Ala102/Phe106 mutant. All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca2+-independent membrane-damaging activities of the recombinant proteins, were evaluated. The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu106→Phe) and L5F/F102A/L106F mutant proteins were identical. The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm. Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker. However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity. This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca2+-independent membrane damage, and implies that substrate binding retains an important role in this process.

scholarly journals Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 418 ◽  
Author(s):  
Minjae Kim ◽  
Jisu Kang ◽  
Yongsoo Kang ◽  
Beom Kang ◽  
EonSeon Jin
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Conformational Change ◽  
Substrate Binding ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
Zeaxanthin Epoxidase ◽  
Substrate Binding Site ◽  
Single Amino Acid Mutation

The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.

Protease Domain Sodium, Calcium, and Zinc Sites in Factor VIIa: Crystal Structures and Kinetic Studies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 122-122 ◽  
Author(s):  
Amy E. Schmidt ◽  
Pooja Shah ◽  
Emily M. Gauthier ◽  
S. Paul Bajaj
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Protein C ◽  
Factor Viia ◽  
Substrate Binding ◽  
Activated Protein C ◽  
Kinetic Studies ◽  
Protease Domain ◽  
Substrate Binding Site ◽  
Hydrolysis Of

Abstract During physiologic coagulation, the factor VIIa (FVIIa)/tissue factor (TF) complex activates FIX and FX. FVIIa consists of a N-terminal γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like (EGF) domains, and a C-terminal serine protease domain. We obtained crystals of FVIIa/soluble TF in the presence of Na+, Rb+, or Choline+ (Ch+) under conditions containing micromolar concentrations of Zn2+. Rb+ is a large monovalent ion and has been used to identify Na+-sites in several proteins; whereas, Ch+ cannot substitute for Na+. The various crystals diffracted from 2.0 to 2.4 Å and belonged to the space group P212121. In the crystal structures, Na+ or Rb+ in FVIIa coordinates to the carbonyl groups of residues 185 (chymotrypsin numbering), 185A, 221, and 224 as well as to two water molecules. Thus, the Na+-site in FVIIa is similar to that of FXa and activated protein C but not to that of thrombin. Ca2+ in the protease domain of FVIIa is coordinated to the carboxylates of Glu70 and Glu80 as seen earlier by Banner and coworkers. Additionally, the crystal structures also showed two Zn2+-sites, one involving His71 and the other involving His117. The Zn2+-sites are unique to FVIIa since the His residues are not present in other proteases. To investigate the role of Na+, Ca2+, and Zn2+-sites in the protease domain of FVIIa, a series of biochemical and kinetic studies were performed. Na+ increased the kcat for hydrolysis of S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) ~22-fold by FVIIaWT whereas Ca2+ increased it ~by 230-fold. In the presence of Ca2+, Na+ had virtually no effect on the hydrolysis of S-2288; however, in the presence of Na+, Ca2+ increased the kcat ~12-fold. Thus, the increase in kcat by Ca2+ in the presence or absence of Na+ was similar (~250-fold). Further, Na+ had no effect on Km whereas Ca2+ increased it ~3.5-fold. However, the increase by in Km is biologically not pertinent since the Gla and EGF1 domains of FVIIa determine the Km for activation of FIX and FX. Moreover, FVIIaF225P (Na+-site mutant) showed little response to Na+ and FVIIaE80V (Ca2+-site mutant) showed no response to Ca2+ in hydrolyzing S-2288. These data indicate that the Na+ and Ca2+ effects observed are due to the occupancy of the protease domain Na+ and Ca2+ sites. Consistent with the Km data, Na+ had no effect on the binding of p-aminobenzamidine (pAB, S1 site probe) to FVIIaWT. Interestingly, Ca2+ decreased the Ki for pAB binding by ~5-fold indicating that the increase in Km for S-2288 caused by Ca2+ is not related to the S1 site but rather to the S2 and/or S3/S4 sites in FVIIa. In further studies, Zn2+ inhibited the potentiation of S-2288 hydrolysis by FVIIaWT with Ki ~1 of μM in the absence and ~30 μM in the presence of Ca2+. We conclude that the Na+-site in FVIIa is not linked to the synthetic substrate binding site(s), and that the Ca2+-site is linked to the substrate binding site(s). These observations are in contrast to what has been previously observed for FXa and activated protein C. Thus, in the absence of TF, Na+ and Ca2+ are positive regulators for catalysis by FVIIa; whereas, Zn2+ exerts a negative effect. Conceivably, occupancy of the Na+-site and the protease domain Ca2+-site may render FVIIa in a conformation suitable for TF binding and substrate hydrolysis. The local Zn2+ concentration following release by activated platelets at the site of hemostasis could dampen coagulation as a regulatory mechanism.

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF DOMAINS IN THE HEAVY CHAIN REGION OF FACTOR XI (XIa) INVOLVED IN BINDING HIGH MOLECULAR WEIGHT KININOGEN AND FACTOR IX

1987 ◽  
Author(s):  
F A Baglia ◽  
D Sinha ◽  
P N Walsh
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Binding Site ◽  
High Molecular Weight ◽  
Heavy Chain ◽  
Substrate Binding ◽  
The Other ◽  
High Molecular Weight Kininogen ◽  
Factor Xi ◽  
Substrate Binding Site

Previous studies from our laboratory (J. Biol. Chem. 260:10714,1985; J. Clin. Invest. 78:1631,1986) provide evidence that a monoclonal antibody (3C1) directed against the heavy chain region of factor XIa (FXIa) recognizes an epitope near a substrate binding site for FIX and a binding site for high molecular weight kininogen (HMWK). The present studies were carried out to determine whether these two sites are identical or different. Another heavy-chain-specific murine monoclonal antibody (5F7) was found to recognize an epitope distinct from that recognized by 3C1 since 3C1 did not compete with 5F7 for binding to FXI in a solid-phase radioimmunoassay. Antibody 3C1 was a competitive inhibitor of F-XIa-catalyzed F-IX activation, assayed by the release of a 3H-labeled activation peptide from FIX, whereas 5F7 had no effect on F—IX activation by FXIa. In contrast, 5F7 (which also inhibited F-XIIa-catalyzed F-XI activation in the presence of HMWK and kaolin) completely blocked FXI binding to immobilized HMWK at concentrations 1,000-fold lower than 3C1. Finally, HMWK had no effect on F-IX activation by FXIa. We therefore conclude that two separate and distinct domains are present in the heavy-chain region of FXI, one of which is a substrate binding site for FIX and the other a binding site for HMWK. A 15,000 Mr peptide containing the HMWK binding site was isolated using cyanogen bromide digests of factor XI which were bound to and eluted from a ,5F7 antibody affinity column and further purified using high performance liquid chromatography. Gas phase sequencing studies are in progress to characterize this peptide and place its sequence within the known structure of the heavy chain of FXIa. In conclusion, our antibodies have defined two domains within the heavy chain region of FXI: one defined by 5F7 is near the HMWK binding site, whereas the other, recognized by 3C1, is a substrate binding site for FIX. Finally, a peptide domain in the heavy chain of FXI that compriaes the HMWK binding site has been identified and isolated.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

2020 ◽  
Vol 21 (2) ◽  
pp. 117-130 ◽  
Author(s):  
Mohammad J. Hosen ◽  
Mahmudul Hasan ◽  
Sourav Chakraborty ◽  
Ruhshan A. Abir ◽  
Abdullah Zubaer ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Glucose Transporter ◽  
Substrate Binding ◽  
Mutation Screening ◽  
Homology Model ◽  
Connective Tissue Disorder ◽  
Crystallographic Structure ◽  
Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

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