Identification of Amino Acid Residues of the Retroviral Aspartic Proteinases Important for Substrate Specificity and Catalytic Efficiency

ABSTRACT Lysine decarboxylase (LDC; EC 4.1.1.18 ) from Selenomonas ruminantium comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both l-lysine andl-ornithine with similar Km andVmax values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063–1069, 1999). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17 ), including the mouse,Saccharomyces cerevisiae, Neurospora crassa,Trypanosoma brucei, and Caenorhabditis elegansenzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved inS. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward bothl-lysine and l-ornithine with a substrate specificity ratio of 0.83 (defined as thek cat/Km ratio obtained with l-ornithine relative to that obtained withl-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.

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Amino Acid Residues Involved in the Substrate Specificity of TauT/SLC6A6 for Taurine and γ-Aminobutyric Acid

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b13-00991 ◽

2014 ◽

Vol 37 (5) ◽

pp. 817-825 ◽

Cited By ~ 6

Author(s):

Tohru Yahara ◽

Masanori Tachikawa ◽

Shin-ichi Akanuma ◽

Yoshiyuki Kubo ◽

Ken-ichi Hosoya

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Aminobutyric Acid ◽

Amino Acid Residues

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On the chemical diversity of the prebiotic ocean of early Earth

International Journal of Astrobiology ◽

10.1017/s1473550414000688 ◽

2014 ◽

Vol 14 (3) ◽

pp. 497-504

Author(s):

Carlo Canepa

Keyword(s):

Amino Acid ◽

Average Length ◽

Chemical Species ◽

Catalytic Efficiency ◽

Chemical Diversity ◽

Aqueous Environment ◽

Amino Acid Residues ◽

Catalytically Active ◽

Order Of Magnitude ◽

The Mean

AbstractThis work investigates the consequences on the diverse number of chemical species in a pre-biotic terrestrial aqueous environment endowed with an amino acid source induced by the spontaneous build-up of catalytically active polypeptides from amino acid monomers. The assumed probability that a randomly formed polypeptide exhibits catalytic properties is dependent on constraining both the chemical identity and the position of a fraction of the amino acid residues. Within this hypothesis, and using values of the average length n of the catalytic polypeptides about one half of the present-day enzymes, the stationary-state concentration of the catalytically active polypeptides is ≈10−30 −10−19 M, and the ratio of the concentration of a product of a catalytic process to the initial concentration of the corresponding substrate is predicted to be ≈10−6−105. Matching the mean life of each catalytic polypeptide to the mean life of its substrate (λ ≈ ω) is only possible by significantly raising the intensity of the source of the amino acid monomers. Under these hypothetical optimal conditions, the mean lives of the catalytic polypeptides and their substrates have values ω−1 ≈ λ−1 ≈10 yr and the asymptotic concentration of each product is of the same order of magnitude as the concentration of the substrate. In all cases the catalytic efficiency necessary to form the active peptides takes the typical values of present-day enzymes.

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Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p inSaccharomyces cerevisiae

Yeast ◽

10.1002/yea.792 ◽

2001 ◽

Vol 18 (15) ◽

pp. 1429-1440 ◽

Cited By ~ 15

Author(s):

Birgitte Regenberg ◽

Morten C. Kielland-Brandt

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Amino Acid Residues ◽

Amino Acid Permeases

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Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

The Journal of Lipid Research ◽

10.1194/jlr.m064121 ◽

2015 ◽

Vol 57 (1) ◽

pp. 89-99 ◽

Cited By ~ 16

Author(s):

Kenshi Watanabe ◽

Makoto Ohno ◽

Masahiro Taguchi ◽

Seiji Kawamoto ◽

Kazuhisa Ono ◽

...

Keyword(s):

Fatty Acid ◽

Amino Acid ◽

Substrate Specificity ◽

Amino Acid Residues ◽

Fatty Acid Desaturases ◽

Front End ◽

Membrane Bound

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Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

Applied and Environmental Microbiology ◽

10.1128/aem.68.6.2676-2682.2002 ◽

2002 ◽

Vol 68 (6) ◽

pp. 2676-2682 ◽

Cited By ~ 32

Author(s):

Daohai Zhang ◽

Xianzhen Li ◽

Lian-Hui Zhang

Keyword(s):

Amino Acid ◽

Sequence Alignment ◽

Half Life ◽

Bacterial Isolate ◽

Catalytic Efficiency ◽

Fold Increase ◽

Maximum Activity ◽

Amino Acid Residues ◽

Sequence Domain ◽

Gene Cloning And Characterization

ABSTRACT The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)8-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp241, Glu295, Asp369, His145, and His368) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a Km of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe3+ and Hg2+ and was enhanced by Mn2+ and Mg2+. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu498 and Arg310 with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.

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Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes

Protein Science ◽

10.1110/ps.ps.25801 ◽

2001 ◽

Vol 10 (12) ◽

pp. 2439-2450 ◽

Cited By ~ 66

Author(s):

N. S. Andreeva

Keyword(s):

Amino Acid ◽

Crystal Structures ◽

Catalytic Site ◽

Amino Acid Residues ◽

Aspartic Proteinases

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The Specific Contribution of Amino Acids 334 and 335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase.

Blood ◽

10.1182/blood.v106.11.1027.1027 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1027-1027

Author(s):

Melissa A. Blum ◽

Tivadar Orban ◽

Daniel O. Beck ◽

Michael Kalafatis

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Heavy Chain ◽

Factor Xa ◽

Catalytic Efficiency ◽

Amino Acid Residues ◽

Wild Type ◽

Factor Va ◽

Cofactor Activity ◽

Type Factor

Abstract The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.

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