scholarly journals Mutational Analysis of Substrate Interactions with the Active Site of Dialkylglycine Decarboxylase

Biochemistry ◽  
2010 ◽  
Vol 49 (30) ◽  
pp. 6485-6493 ◽  
Author(s):  
Emily J. Fogle ◽  
Michael D. Toney
Keyword(s):  
Active Site ◽  
Mutational Analysis ◽  
Substrate Interactions ◽  
Dialkylglycine Decarboxylase

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals A Mutational Analysis of the Active Site Loop Residues incis-3-Chloroacrylic Acid Dehalogenase

Biochemistry ◽  
2013 ◽  
Vol 52 (24) ◽  
pp. 4204-4216 ◽  
Author(s):  
Gottfried K. Schroeder ◽  
Jamison P. Huddleston ◽  
William H. Johnson ◽  
Christian P. Whitman
Keyword(s):  
Active Site ◽  
Mutational Analysis ◽  
Chloroacrylic Acid Dehalogenase

Catalytic Mechanism of SHCHC Synthase in the Menaquinone Biosynthesis ofEscherichia coli: Identification and Mutational Analysis of the Active Site Residues

Biochemistry ◽  
2009 ◽  
Vol 48 (29) ◽  
pp. 6921-6931 ◽  
Author(s):  
Ming Jiang ◽  
Xiaolei Chen ◽  
Xian-Hui Wu ◽  
Minjiao Chen ◽  
Yun-Dong Wu ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Ofescherichia Coli ◽  
Active Site Residues

Structure, interactions and action of Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase

2018 ◽  
Vol 475 (15) ◽  
pp. 2457-2471 ◽  
Author(s):  
Rajapiramuthu Srikalaivani ◽  
Amrita Singh ◽  
Mamannamana Vijayan ◽  
Avadhesha Surolia
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Mutational Analysis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Native Page ◽  
Acid Dehydrogenase ◽  
Binary Complexes ◽  
Solution Studies ◽  
Hydroxyisobutyric Acid

Biochemical and crystallographic studies on Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase (MtHIBADH), a member of the 3-hydroxyacid dehydrogenase superfamily, have been carried out. Gel filtration and blue native PAGE of MtHIBADH show that the enzyme is a dimer. The enzyme preferentially uses NAD+ as the cofactor and is specific to S-hydroxyisobutyric acid (HIBA). It can also use R-HIBA, l-serine and 3-hydroxypropanoic acid (3-HP) as substrates, but with much less efficiency. The pH optimum for activity is ∼11. Structures of the native enzyme, the holoenzyme, binary complexes with NAD+, S-HIBA, R-HIBA, l-serine and 3-HP and ternary complexes involving the substrates and NAD+ have been determined. None of the already known structures of HIBADH contain a substrate molecule at the binding site. The structures reported here provide for the first time, among other things, a clear indication of the location and interactions of the substrates at the active site. They also define the entrance of the substrates to the active site region. The structures provide information on the role of specific residues at the active site and the entrance. The results obtained from crystal structures are consistent with solution studies including mutational analysis. They lead to the proposal of a plausible mechanism of the action of the enzyme.

The Roles of Active-Site Residues in the Catalytic Mechanism oftrans-3-Chloroacrylic Acid Dehalogenase:  A Kinetic, NMR, and Mutational Analysis†

Biochemistry ◽  
2004 ◽  
Vol 43 (14) ◽  
pp. 4082-4091 ◽  
Author(s):  
Hugo F. Azurmendi ◽  
Susan C. Wang ◽  
Michael A. Massiah ◽  
Gerrit J. Poelarends ◽  
Christian P. Whitman ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Active Site Residues ◽  
Chloroacrylic Acid Dehalogenase

scholarly journals Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

2001 ◽  
Vol 358 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Henning B. BOLDT ◽  
Michael T. OVERGAARD ◽  
Lisbeth S. LAURSEN ◽  
Kathrin WEYER ◽  
Lars SOTTRUP-JENSEN ◽  
...  
Keyword(s):  
Plasma Protein ◽  
Active Site ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Protein A ◽  
Residual Activity ◽  
Zinc Binding ◽  
Binding Motif ◽  
Dependent Manner ◽  
Zinc Binding Motif

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

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