Kinetic mechanism of the interaction of D-cycloserine with serine hydroxymethyltransferase

The kinetic mechanism for the interaction of D-cycloserine with serine hydroxymethyltransferase (EC2.1.2.1) from sheep liver was established by measuring changes in the activity, absorbance, and circular dichoism (CD) of the enzyme. The irreversible inhibition of the enzyme was characterized by three detectable steps: an initial rapid step followed by two successive steps with rate constants of 5.4 X s-l and 1.4 X lo4 s-l. The first step was distinguished by a rapid disappearance of the enzyme absorbance peak at 425 nm, a decrease in the enzyme activity to 25% of the uninhibited velocity, and a lowering of the CD intensity at 432 nm to about 65% of the original value. The second step of the interaction was accompanied by a complete loss of enzyme

A hemagglutinin from Pongamia pinnata (L.) Pierre seed

397-403Lectins are diverse proteins that bind to carbohydrates and are involved in many important physiological processes. They are highly specific to the sugar molecules they bind and therefore, have many therapeutic and diagnostic applications. There are several lectins that display antiviral, antibacterial antifungal and antitumour activities. Characterization of new lectins paves way for comprehension of their diverse biological roles and mechanism of action, thus aiding in further exploration of lectins in various domains of biology. Here, we endeavoured to purify and characterize lectin from the seed of the legume,Pongamia. Pongamia pinnata (L.) Pierre (syn. Millettia pinnata), seed lectin (PPSL) was purified conventionally by ammonium-sulfate precipitation followed by size exclusion chromatography. The further lectin was physicochemically characterized by CD, fluorescence spectroscopy, mass spectroscopy and isothermal calorimetry. Hemagglutination studies with various mono and disaccharides showed specificity towards galactose. This specificity was reaffirmed by isothermal studies with appreciable thermodynamic parameters. Lectins have tremendous diagnostic applications. They are used as second-generation drug delivery systems

A hemagglutinin from Pongamia pinnata (L.) Pierre seed

Lectins are diverse proteins that bind to carbohydrates and are involved in many important physiological processes. They are highly specific to the sugar molecules they bind and therefore, have many therapeutic and diagnostic applications. There are several lectins that display antiviral, antibacterial antifungal and antitumour activities. Characterization of new lectins paves way for comprehension of their diverse biological roles and mechanism of action, thus aiding in further exploration of lectins in various domains of biology. Here, we endeavoured to purify and characterize lectin from the seed of the legume,Pongamia. Pongamia pinnata (L.) Pierre (syn. Millettia pinnata), seed lectin (PPSL) was purified conventionally by ammonium-sulfate precipitation followed by size exclusion chromatography. The further lectin was physicochemically characterized by CD, fluorescence spectroscopy, mass spectroscopy and isothermal calorimetry. Hemagglutination studies with various mono and disaccharides showed specificity towards galactose. This specificity was reaffirmed by isothermal studies with appreciable thermodynamic parameters. Lectins have tremendous diagnostic applications. They are used as second-generation drug delivery systems

The role of inter-subunit ionic interactions in the sssembly of Physalis mottle tymovirus

Physalis mottle tymovirus (PhMV) is a small spherical plant virus with its RNA genome encapsidated in a protein shell made of 180 identical coat protein (CP) subunits. The amino acid residues involved in two interfacial salt bridges, Asp-83/Arg-159 and Arg-68/Asp-150 and Lys-153, were targeted for mutagenesis with a view to delineate the role of interfacial ionic interactions in the subunit folding and assembly of the virus. R159A and D83A-R159A recombinant CP (rCP) mutants formed stable T=3 capsids, indicating that the D83-R159 interfacial salt bridge is dispensable for the folding and assembly of PhMV. However, D150A and R68Q-D150A mutant rCPs were present in the insoluble fraction, suggesting that the R68-D150 interfacial salt bridge is crucial for subunit folding and assembly. Similarly, K153Q, D83A-K153Q, and H69AK153Q mutant rCPs were present in the insoluble fraction. Interestingly, the R68Q-D150A, D83A-K153Q, and H69A-K153Q double mutant rCPs could be refolded into partially folded soluble heterogeneous aggregates of 14–16 S. The results further confirm our earlier observation that subunit folding and assembly are concerted events in PhMV

Mechanism of interaction of O-amino-D-serine with sheep liver serine hydroxymethyltransferase

The mechanism of interaction of 0-amino-D-serine (OADS) with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in the enzyme activity,absorption spectra, circular dichroism (CD) spectra, and stopped-flow spectrophotometry. OADS was a reversible noncompetitive inhibitor (Ki = 1.8 pM) when serine was the varied substrate. The first step in the interaction of OADS with the enzyme was the disruption of enzyme-Schiff base, characterized by the rapid disappearance of absorbance at 425 nm (6.5 X lo3 M-' s-') and CD intensity at 430 nm. Concomitantly,there was a rapid increase in absorbance and CD intensity at 390 nm. The spectral properties of this intermediate enabled its identification as pyridoxal 5'-phosphate (PLP). These changes were followed by a slow unimolecular step (2 X s-') leading to the formation of PLP-OADS oxime, which was confirmed by its absorbance and fluorescence spectra and retention time on high-performance liquid chromatography. The PLP-OADS oxime was displaced from the enzyme by the addition of PLP as evidenced by the restoration of complete enzyme activity as well as by the spectral properties. The unique feature of the mechanism proposed for the interaction of OADS with sheep liver SHMT was the formation of PLP as an intermediate

Napin from Brassica juncea: Thermodynamic and structural analysis of stability

The napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits – one small (29 amino acid residues) and one large (86 amino acids residues) – held together by disulfide bonds. The thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with $T_{M1}$ and $T_{M2}$ at 323.5 K and 335.8 K, respectively; $\Delta C_{P1}$ and $\Delta C_{P2}$ are $2.05 kcal mol^{-1} K^{-1}$ and $1.40 kcal mol^{−1} K^{−1}$, respectively. In the temperature range 310–318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N⇆I⇆U with $\Delta G_{1H2O}$ and $\Delta G_{2H2O}$ values of $5.2 kcal mol^{−1}$ and $5.1 kcal mol^{−1}$ at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from $20 \AA$ to $35.0 \AA$ due to unfolding by guanidinium hydrochloride. In silico alignment of sequences of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition

Molecular Mechanism of Dimerization of Bowman-Birk Inhibitors: PIVOTAL ROLE OF ASP76ASP^{76} IN THE DIMERZATION

Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, is a crop native to Southeast Asia and tropical Africa. The seeds contain multiple forms of Bowman- Birk type inhibitors. The major inhibitor HGI-III, from the native seed with 76 amino acid residues exists as a dimer. The amino acid sequence of three isoforms of Bowman-Birk inhibitor from germinated horsegram, designated as HGGI-I, HGGI-II, and HGGI-III, have been obtained by sequential Edman analyses of the pyridylethylated inhibitors and peptides derived therefrom by enzymatic and chemical cleavage. The HGGIs are monomers, comprising of 66, 65, and 60 amino acid residues, respectively. HGGI-III from the germinated seed differs from the native seed inhibitor in the physiological deletion of a dodecapeptide at the amino terminus and a tetrapeptide, -SHDD, at the carboxyl terminus. The study of the state of association of HGI-III, by size-exclusion chromatography and SDS-PAGE in the presence of 1 mM $ZnCl_2$, has revealed the role of charged interactions in the monomer $\leftrightarrow$ dimer equilibria. Chemical modification studies of Lys and Arg have confirmed the role of charge interactions in the above equilibria. These results support the premise that a unique interaction, which stabilizes the dimer, is the cause of selfassociation in the inhibitors. This interaction in HGI-III involves the $\in$-amino group of the $Lys^{24}$ $(P_1$ residue) at the first reactive site of one monomer and the carboxyl of an $Asp^{76}$ at the carboxyl terminus of the second monomer.Identification of the role of these individual amino acids in the structure and stability of the dimmer was accomplished by chemical modifications, multiple sequence alignment of legume Bowman-Birk inhibitors, and homology modeling. The state of association may also influence the physiological and functional role of these inhibitors