Side-Chain Conformational Thermodynamics of Aspartic Acid Residue in the Peptides and Achatin-I in Aqueous Solution

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

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Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position

Biochemistry ◽

10.1021/bi00080a026 ◽

1993 ◽

Vol 32 (29) ◽

pp. 7526-7530 ◽

Cited By ~ 23

Author(s):

John E. Ladbury ◽

Richard Wynn ◽

Homme W. Hellinga ◽

Julian M. Sturtevant

Keyword(s):

Escherichia Coli ◽

Aspartic Acid ◽

Aspartic Acid Residue

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NMR Study of Amino Acids and Their Derivatives. V. Structures and Formation Constants of Zinc-L-Aspartic Acid Complexes in Aqueous Solution

Bulletin of the Chemical Society of Japan ◽

10.1246/bcsj.46.468 ◽

1973 ◽

Vol 46 (2) ◽

pp. 468-471 ◽

Cited By ~ 18

Author(s):

Hidehiro Ishizuka ◽

Takeo Yamamoto ◽

Yoji Arata ◽

Shizuo Fujiwara

Keyword(s):

Aqueous Solution ◽

Amino Acids ◽

Aspartic Acid ◽

Formation Constants ◽

Nmr Study

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Amino acids important in enzyme activity and dimer stability for Drosophila alcohol dehydrogenase

Biochemical Journal ◽

10.1042/bj3080419 ◽

1995 ◽

Vol 308 (2) ◽

pp. 419-423 ◽

Cited By ~ 12

Author(s):

S W Chenevert ◽

N G Fossett ◽

S H Chang ◽

I Tsigelny ◽

M E Baker ◽

...

Keyword(s):

Enzyme Activity ◽

Alcohol Dehydrogenase ◽

Binding Site ◽

Aspartic Acid ◽

Three Dimensional ◽

Side Chain ◽

Wild Type ◽

Dimensional Model ◽

Three Dimensional Model ◽

Substrate Binding Site

We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.

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