scholarly journals Evolution of Substrate Specificity within a Diverse Family of β/α-Barrel-fold Basic Amino Acid Decarboxylases

2010 ◽  
Vol 285 (33) ◽  
pp. 25708-25719 ◽  
Author(s):  
Xiaoyi Deng ◽  
Jeongmi Lee ◽  
Anthony J. Michael ◽  
Diana R. Tomchick ◽  
Elizabeth J. Goldsmith ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Basic Amino Acid

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

2008 ◽  
Vol 416 (1) ◽  
pp. 109-116 ◽  
Author(s):  
Yoshikuni Goto ◽  
Hiroe Tanji ◽  
Akira Hattori ◽  
Masafumi Tsujimoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Enzymatic Activity ◽  
Basic Amino Acid ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Peptide Substrates ◽  
Endoplasmic Reticulum Aminopeptidase 1 ◽  
Natural Peptides

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln181 as important for enzymatic activity and substrate specificity. Replacement of Gln181 by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln181 is critical for the enzymatic activity and substrate specificity of ERAP-1.

scholarly journals Phylogenetic Diversity and the Structural Basis of Substrate Specificity in the β/α-Barrel Fold Basic Amino Acid Decarboxylases

2007 ◽  
Vol 282 (37) ◽  
pp. 27115-27125 ◽  
Author(s):  
Jeongmi Lee ◽  
Anthony J. Michael ◽  
Dariusz Martynowski ◽  
Elizabeth J. Goldsmith ◽  
Margaret A. Phillips
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Phylogenetic Diversity ◽  
Basic Amino Acid ◽  
Structural Basis

Series of sequential polypeptides containing lysine or arginine: Preparation and characterization

1988 ◽  
Vol 53 (1) ◽  
pp. 145-156 ◽  
Author(s):  
Jana Pírková ◽  
Svetlana Churkina ◽  
Vladimír Gut ◽  
Ivo Frič ◽  
Karel Bláha
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Synthesis ◽  
Average Molecular Weight ◽  
Basic Amino Acid ◽  
Cd Spectra ◽  
Active Esters ◽  
Marked Tendency

The sequential polypeptides (Lys-Ala)n, (Lys-Ala-Ala)n, (Lys-Ala-Ala-Ala)n, (Lys-Leu-Ala)n, (Lys-Leu-Ala-Ala)n, (Lys-Leu-Ala-Ala-Ala)n, (Lys-Ala-Leu)n, (Lys-Ala-Leu-Ala)n, (Orn-Leu-Ala)n,(Arg-Ala-Ala)n, (Arg-Leu-Ala)n, (Arg-Leu-Ala-Ala)n, (Arg-Ala-Leu)n, and (Arg-Ala-Leu-Ala)n were synthesized by polymerization of active esters (1-succinimidyl or pentafluorophenyl) of the corresponding Nα-deblocked monomers. The monomers were prepared using the usual methods of peptide synthesis in solution. Upon dialysis, the average molecular weight of the polymer was 6 000-9 000 as determined by sedimentation in ultracentrifuge. Polypeptides, containing leucine in addition to the basic amino acid, showed a marked tendency to aggregation. CD spectra of the products were measured for characterization.

Comparison of the Potency of 8-L-Arginine, 8-D-Arginine and 8-D-Homoarginine Vasopressin Analogs with Substituted Phenylalanine in Position 2

1994 ◽  
Vol 59 (6) ◽  
pp. 1439-1450 ◽  
Author(s):  
Miroslava Žertová ◽  
Jiřina Slaninová ◽  
Zdenko Procházka
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Basic Amino Acid ◽  
The Other ◽  
Side Chain ◽  
Inhibitory Potency ◽  
Phase Synthesis ◽  
Other Hand ◽  
Order Of Magnitude

An analysis of the uterotonic potencies of all analogs having substituted L- or D-tyrosine or -phenylalanine in position 2 and L-arginine, D-arginine or D-homoarginine in position 8 was made. The series of analogs already published was completed by the solid phase synthesis of ten new analogs having L- or D-Phe, L- or D-Phe(2-Et), L- or D-Phe(2,4,6-triMe) or D-Tyr(Me) in position 2 and either L- or D-arginine in position 8. All newly synthesized analogs were found to be uterotonic inhibitors. Deamination increases both the agonistic and antagonistic potency. In the case of phenylalanine analogs the change of configuration from L to D in position 2 enhances the uterotonic inhibition for more than 1 order of magnitude. The L to D change in position 8 enhances the inhibitory potency negligibly. Prolongation of the side chain of the D-basic amino acid in position 8 seems to decrease slightly the inhibitory potency if there is L-substituted amino acid in position 2. On the other hand there is a tendency to the increase of the inhibitory potency if there is D-substituted amino acid in position 2.

scholarly journals Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

1988 ◽  
Vol 251 (3) ◽  
pp. 691-699 ◽  
Author(s):  
R W Olafson ◽  
W D McCubbin ◽  
C M Kay
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Helical Structure ◽  
Basic Amino Acid ◽  
Cysteine Residues ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Empirical Relations ◽  
Heavy Metal Binding ◽  
Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

scholarly journals Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity.

1987 ◽  
Vol 262 (8) ◽  
pp. 3754-3761
Author(s):  
A.J. Ganzhorn ◽  
D.W. Green ◽  
A.D. Hershey ◽  
R.M. Gould ◽  
B.V. Plapp
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue ◽  
Kinetic Characterization ◽  
Alcohol Dehydrogenases

Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis

2021 ◽  
Author(s):  
Ryushi Kawakami ◽  
Chinatsu Kinoshita ◽  
Tomoki Kawase ◽  
Mikio Sato ◽  
Junji Hayashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Enzyme Stability ◽  
Hyperthermophilic Archaea ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
Aminobutyrate Aminotransferase ◽  
Chaperone Protein ◽  
Amino Acid Racemase

Abstract The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

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