scholarly journals Ionic interactions in the formation of the thrombin-hirudin complex

1991 ◽  
Vol 275 (3) ◽  
pp. 801-803 ◽  
Author(s):  
A Betz ◽  
J Hofsteenge ◽  
S R Stone
Keyword(s):  
Amino Acids ◽  
Binding Energy ◽  
Glutamic Acid ◽  
Crystal Structures ◽  
Site Directed Mutagenesis ◽  
Ionic Interactions ◽  
Directed Mutagenesis ◽  
Salt Bridges ◽  
Acidic Residues ◽  
Terminal Amino

Site-directed mutagenesis has been used to examine the importance of each of the acidic C-terminal residues of hirudin in the formation of its complex with alpha-thrombin. The contribution to binding energy of acidic residues in the 11 C-terminal amino acids varied from 2.3 to 5.9 kJ.mol-1. The differences between the contributions of individual residues were smaller than would be expected from the crystal structures of the thrombin-hirudin complex. In particular, the small effect (2.4 kJ.mol-1) for the replacement of Asp-55 was surprising in view of the two salt bridges made by this residue. The results of studies involving multiple mutations indicated that the additivity of the effects varied with the position of the mutation. Whereas the effect of mutations involving the glutamic acid residues at positions 61 and 62 were additive, non-additivity was observed with the glutamic acid residues at positions 57 and 58.

scholarly journals Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans

1994 ◽  
Vol 302 (1) ◽  
pp. 291-295 ◽  
Author(s):  
A Moreau ◽  
M Roberge ◽  
C Manin ◽  
F Shareck ◽  
D Kluepfel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzyme Activity ◽  
Streptomyces Lividans ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Protein Mutation ◽  
Acidic Residues

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.

scholarly journals Lysine residues form an integral component of a novel NH2-terminal membrane targeting motif for myristylated pp60v-src.

1992 ◽  
Vol 119 (2) ◽  
pp. 415-425 ◽  
Author(s):  
L Silverman ◽  
M D Resh
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Membrane Receptors ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Lysine Residues ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Contact Mechanism

Association of pp60v-src with the plasma membrane is fundamental to generation of the transformed phenotype. Although myristylation of pp60v-src is required for interaction with a membrane-bound receptor, the importance of NH2-terminal amino acids in receptor binding has not yet been uncoupled from their role in signaling myristylation. Using chimeric src proteins, peptides identical or related to the NH2 terminus of src, and site-directed mutagenesis, we demonstrate that NH2-terminal lysines in conjunction with myristate are essential for membrane localization. Subsequent to NH2-terminal interaction with the "src receptor," internal regions of the src protein also participate in membrane binding. This novel NH2-terminal motif and internal contact mechanism may direct other members of the src family of tyrosine kinases to their membrane receptors.

scholarly journals Contribution of interactions with the core domain of hirudin to the stability of its complex with thrombin

1994 ◽  
Vol 298 (2) ◽  
pp. 507-510 ◽  
Author(s):  
A Betz ◽  
P C R Hopkins ◽  
B F Le Bonniec ◽  
S R Stone
Keyword(s):  
Crystal Structure ◽  
Binding Energy ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Ionic Interactions ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Core Domain ◽  
The Core ◽  
The Stability

The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.

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