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M A Qasim

Publications and source records attributed to M A Qasim.

At least 37 records · Page 2Linked to original sources
PubMed1997

Binding of amino acid side-chains to S1 cavities of serine proteinases.

The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (+/-2) degrees C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1, cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin A alpha (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 10(6) to 10(8). The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu- and Asp- are strongly unfavorable. The Pearson pro duct moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (-0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.

Amino Acid Sequence↗
PubMed1997

Interscaffolding additivity. Association of P1 variants of eglin c and of turkey ovomucoid third domain with serine proteinases.

Standard mechanism protein inhibitors of serine proteinases share a common mechanism of interaction with their cognate enzymes. The P1 residue of the inhibitor interacts with the enzyme in a substrate-like manner. Its side chain becomes imbedded in the S1 cavity of the enzyme. The nature of P1, the primary specificity residue, greatly affects the strength and specificity of the enzyme inhibitor association. In canonical inhibitors, residues P4-P2'(P3'), where P1-P1' is the reactive site, share a common main chain conformation that does not change on complex formation. The remainder of the inhibitor's structure, the scaffolding, is not always common. Instead, there are at least 20 inhibitor families, each with a different scaffolding. In this paper, we ask whether the differences in standard free energy of association of enzyme-inhibitor complexes upon P1 mutations are independent of the nature of the scaffolding. We have already reported on 25 P1 variants of turkey ovomucoid third domain, a member of the Kazal inhibitor family, interacting with six different serine proteinases. Here, we report on seven different P1 variants of eglin c, a potato I family member, interacting with the same six serine proteinases under the same conditions. The differences in standard free energy on P1 mutations in the eglin c system agree very well, when P1 Pro is omitted. Complete agreement indicates that these P1 residues are interscaffolding additive. This is consistent with the superimposition of the high-resolution structures of eglin c and of turkey ovomucoid third domain with chymotrypsin. In both cases, the P1 Leu side chain is similarly oriented in almost indistinguishable specificity pockets of the enzyme.

Amino Acid Sequence↗
PubMed1997

Probing intermolecular main chain hydrogen bonding in serine proteinase-protein inhibitor complexes: chemical synthesis of backbone-engineered turkey ovomucoid third domain.

Intermolecular main chain H-bonding networks are frequently encountered at the interface of complexes of protein proteinase inhibitors and their cognate enzymes. Studies of X-ray crystal structures of many protein inhibitors complexed with serine proteinases have revealed that the amide NH group of the P1 residue in the inhibitor donates an H-bond to the carbonyl C = O group of Ser214 and Ser195 Oy in the enzyme (Ser125 and Ser221 in subtilisins, respectively). To probe the energetic contribution of this backbone H-bond in the complexes of OMTKY3 with several serine proteinases, native chemical ligation was used for the total synthesis of a backbone-engineered analog of OMTKY3, in which the amide peptide bond between Thr17 (P2) and Leu18 (P1) was replaced by an ester bond, i.e., -CONH-to-COO-. This chemical "mutation" effectively eliminated the backbone H-bond donated by the NH group of Leu18. By measuring association equilibrium constants for synthetic wild-type OMTKY3 and the backbone-engineered ester analog interacting with a panel of six serine proteinases, we have determined that the P1 NH-->O substitution weakens the binding of OMTKY3 to its cognate enzymes by an average of 15-fold, i.e., 1.5 +/- 0.3 kcal/mol. These results place a quantitative value on the contribution of the intermolecular backbone H-bond in enzyme-inhibitor recognition.

Amino Acid Sequence↗
PubMed1997

Interaction of bromocresol green with different serum albumins studied by fluorescence quenching.

The binding of bromocresol green to bovine serum albumin at micromolar concentrations leads to quenching of protein fluorescence. This property has been used here to study interaction of bromocresol green with bovine serum albumin as a function of pH and ionic strength. The transformation of fluorescence quench data obtained with bromocresol green into Scatchard plots yielded an association constant of 3.06 x 10(7) 1 M-1 and a binding capacity of about 1.0. The affinity of bromocresol green for bovine serum albumin remains virtually unchanged between pH 4.0 and 8.0 but decreases by about 7 fold with increase in ionic strength from 0.01 to 1.0. Six other serum albumins obtained from cat, dog, human, pig and sheep have also been studied for bromocresol green binding. Although all the albumins studied bind bromocresol green, they show considerable differences in their affinities towards the dye. It appears that despite a great degree of overall similarity in their structure and conformation, serum albumins from different species differ in their ligand binding properties.

Animals↗
PubMed1996

Isolation and characterization of domain I of ovoinhibitor.

Domain I of ovoinhibitor was isolated by subjecting the protein to specific chemical cleavage by cyanogen bromide followed by repeated gel filtration. The first domain of ovoinhibitor was found to be homogeneous by the criteria of gel chromatography, SDS-PAGE and PAGE. Mr values by gel filtration (10900) and SDS-PAGE (8300) were slightly higher than that computed from amino-acid sequence. This discrepancy has been attributed to the glycoprotein nature of domain I as it was found to contain 10% neutral carbohydrate and 2% sialic acid. Fluorescence spectral properties showed the presence of tryptophan in domain I. The amino-acid composition of domain I isolated in this study was in very good agreement with that computed from amino-acid sequence. Gel filtration behaviour of the first domain was consistent with a Stokes radius of 1.6 nm and a frictional ratio of 1.2 suggesting asymmetry and/or excessive hydration. Domain I was found to be a potent inhibitor of bovine trypsin but was virtually devoid of activity against chymotrypsin, elastase and proteinase K. The equilibrium association constant for domain I-trypsin complex was computed to be 6.6x10(8)M-1.

Amino Acid Sequence↗
PubMed1995

Fatty acid binding to bovine serum albumin prevents formation of intermediate during denaturation.

Urea-induced denaturation of defatted BSA and BSA containing six mol palmitic acid/mol protein (fatted BSA) has been studied by the techniques of UV difference spectroscopy and fluorescence spectroscopy. The notable differences between the two transitions are a shift in the transition of fatted BSA to a higher urea concentration and a three-state denaturation transition in defatted BSA compared to an apparent two-state denaturation transition in fatted BSA. The stable denaturation intermediate in defatted BSA occurs at 4.5-5.0 M urea, a urea concentration at which denaturation in fatted BSA has not yet started. These results are further supported by the difference spectral results obtained at 4.5 M urea in the two albumin preparations. The occurrence of denaturation intermediate only in defatted BSA and the presence of the two strong fatty-acid-binding sites in domain III lead us to conclude that this domain is relatively unstable in the absence of fatty acids and is responsible for the formation of intermediate.

Fatty Acids↗
PubMed1993

Changes in protein stability upon chemical modification of lysine residues of bovine serum albumin by different reagents.

Using acetic anhydride, potassium cyanate and O-methyl isourea six chemically modified derivatives of bovine serum albumin with chemical modification on lysine side chains have been prepared. All the modified preparations were found to be homogeneous with respect to size and charge. Size exclusion chromatography on a calibrated Sephacryl S-300 column revealed that whereas acetylation and carbamylation produce significant increase in Stokes' radius, guanidination produces a slight decrease in Stokes' radius. Stability of native and different modified preparations was studied by following urea induced denaturation using the technique of uv difference spectroscopy and by measuring delta GDH2O and delta delta GD(urea)50% from these data. The order of stability of different preparations was found to be guanidinated > native > carbamylated > acetylated.

Acetic Anhydrides↗
PubMed1990

Amino acid sequences of ovomucoid third domain from 25 additional species of birds.

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowski et al. (1987, Biochemistry 26, 202-221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro14----Ser14 replacement in weka, an Ala14----Thr15 replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile18 and Gly18 at the P1 reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu34) rather than positive (Lys34 or Arg34) amino acid residue at the NH2 terminus of the alpha helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genus Gallus, in the five species genus Syrmaticus, and in the two species genera Aix and Pavo.

Amino Acid Sequence↗
PubMed1990

Binding of bromophenol blue to bovine serum albumin and its succinylated forms.

Interaction of bromophenol blue with bovine serum albumin and its five succinylated forms was studied spectrophotometrically at three different ionic strengths, i.e. 0.04, 0.15 and 1.0 and at two different pH values, namely pH 7.0 and pH 5.0 respectively. Results showed a decrease in bromophenol blue binding on increasing succinylation at low ionic strengths. This decrease was more marked at pH 7.0 than pH 5.0. However, at both the pH values binding returned to a significant degree on increasing the ionic strength to 1.0. Succinylation also caused marked conformational changes at pH 7.0 and ionic strength 0.15 as evidenced by changes in hydrodynamic properties and reduction in antigen-antibody precipitin reaction. However, an increase in ionic strength to 1.0 or decrease in pH to 5.0 caused significant reversal in hydrodynamic parameters. These studies show that lysine residues of bovine serum albumin are not important in bromophenol blue binding.

Bromphenol Blue↗
PubMed1990

Purification and properties of buffalo serum albumin.

Based on a detailed study of the solubility of serum albumin, a procedure for its purification by selective ammonium sulphate precipitation has been developed. Using buffalo serum, first extraneous proteins were precipitated by making the serum 2.26 M saturated with ammonium sulphate at pH 7.0 and then albumin was precipitated from the supernatant at 1.9 M ammonium sulphate concentration at pH 4.2. The overall yield of serum albumin thus isolated was about 55% with a purity of 97%. The protein preparation gave a single nearly symmetrical peak on Sephadex G-100 column and virtually a single band on polyacrylamide gel electrophoresis in the presence and absence of SDS. Buffalo serum albumin has a molecular weight of 69,000 Da. The hydrodynamic properties such as Stoke's radius (3.70 nm), diffusion coefficient (6.03 X 10(-7) cm2/s) and frictional ratio (1.36) obtained by analytical gel chromatography, bilirubin binding characteristics and its interaction with anti-bovine serum albumin antiserum suggest that buffalo serum albumin is very similar to BSA in its molecular properties.

Ammonium Sulfate↗
PubMed1989

Noninvolvement of surface lysine residues of bovine serum albumin in bilirubin binding.

Interaction of bilirubin with bovine serum albumin and its five succinylated forms was studied using fluorescence spectroscopy at two different ionic strengths i.e., 0.15 and 1.0 respectively. Affinity constant was found to be 1.8 x 10(7) litres/mole at 0.15 ionic strength which decreased to 4.4 x 10(6) litres/mole after 87% succinylation. On increasing ionic strength to 1.0, there was a slight decrease in affinity constant for native albumin. However, affinity constant remained same in 55 and 87% modified albumins at high ionic strength. These results suggest noninvolvement of surface lysine residues in bilirubin albumin complex.

Bilirubin↗
PubMed1987

Influence of succinylation of bovine serum albumin on its conformation and bilirubin binding.

In order to investigate the role of lysine residues in the interaction of bilirubin with bovine serum albumin, five succinylated preparations of albumin, namely: 23%, 39%, 49%, 55% and 87%, were prepared, and their conformational and bilirubin-binding properties were studied by the techniques of gel filtration, ultraviolet and visible spectroscopy, and fluorescence quenching. Gel filtration experiments performed at pH 7.0 and ionic strengths 0.15 and 1.0 suggested that the albumin molecule undergoes gradual disorganization with increase in succinylation. The Stokes radius and frictional ratio at ionic strength 0.15 increased from 3.7 nm and 1.36, respectively, for the native protein to 6.3 nm and 2.26 for maximally (87%) succinylated albumin. Interestingly, increase in ionic strength to 1.0 caused significant refolding in succinylated preparations as evidenced by a decrease in Stokes radius and frictional ratio (5.3 nm and 1.90 for 87% succinylated albumin). Progressive succinylation produced a steady decline in the intensity of bilirubin-induced fluorescence quenching, and in the visible spectral changes of the bilirubin-albumin complex at 480 nm. Both of these changes had a good correlation with increase in Stokes radius. Increase in ionic strength to 1.0 produced a significant reversal in these properties. From these results we conclude that probably none of the surface lysine residues is involved in bilirubin-albumin interaction, and that if lysine residues are involved in this interaction they must be buried in the protein interior.

Albumins↗
PubMed1986

Purification and some properties of buffalo liver cathepsin B.

Buffalo liver cathepsin B was isolated by acid extraction, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-Sephadex chromatography and Sephacryl S-300 chromatography. The enzyme preparation was found to be homogeneous by gel filtration and SDS-polyacrylamide gel electrophoresis but could be resolved into two major and four minor protein bands on polyacrylamide gel electrophoresis in the absence of SDS. The enzyme showed catheptic activity against synthetic substrates such as BANA and BAPNA as well as against denatured hemoglobin. Various physico-chemical and enzymatic properties of the enzyme, such as molecular weight, Stokes radius, frictional coefficient, pH optimum, Michaelis constant, and Vmax, were determined. The values of these parameters were 27,500, 2.41 nm, 1.2, 6.5, 2.08 mM, and 42.4 units/mg, respectively. The hydrodynamic properties suggest a compact and globular conformation for this enzyme. Various compounds were tested for their influence on the activity of cathepsin B. Of these compounds, membrane phospholipids were found to increase significantly the activity of this enzyme. This increase in activity could be of physiological importance since the concentration of phospholipids is increased after endocytosis and autophagy.

Animals↗
PubMed1986

A correlation between changes in conformation and molecular properties of bovine serum albumin upon succinylation.

Using succinic anhydride, six succinylated derivatives of bovine serum albumin having percent modification in the range of 23-87% were prepared and their physicochemical and immunological properties were studied. Measurements of Stokes radius, frictional ratio, UV spectra, solvent perturbation, solubility, and immunological cross-reactivity against anti-bovine serum albumin antiserum revealed that the protein undergoes gradual changes in its native conformation with increase in the degree of succinylation. These changes were less marked below 50% modification but became pronounced above 50% modification. However, even the maximally modified preparation (87%) contained a significant amount of folded structure. Interestingly, though the measurements of various molecular properties revealed significant changes in 23-49% modified preparations, the solubility parameters for these preparations which were obtained at high ionic strength were indistinguishable from those of the native protein. The various results taken together suggest that at lower degrees of chemical modification, the conformational changes were produced mainly because of an increase in electrostatic free energy, whereas at higher degrees of modification, steric hindrance in addition to the electrostatic factor seems to make a substantial contribution to the conformational changes in the modified proteins.

Chemical Phenomena↗
PubMed1986

Altered N----F transition in bovine serum albumin dimer and tetramer.

The monomer, dimer and tetramer of bovine serum albumin were separated by gel filtration on a Sephacryl S-300 column. Their purity was established by gel filtration, polyacrylamide gel electrophoresis and SDS polyacrylamide gel electrophoresis. These preparations were identical in bilirubin binding, cross-reactivity against anti-bovine serum albumin antiserum and UV spectral features. However, the three preparations differed in their N----F transition. The mid points of the N----F transitions for monomer, dimer and tetramer were found to be 3.75, 3.60 and 3.40 respectively. We presume that intermolecular interactions and/or alterations in the pK values of carboxyl groups are responsible for increased stability in the case of dimer and tetramer.

Animals↗
PubMed1981

N-(9-Xanthenyl)amino acid N-carboxyanhydrides in the solid-phase synthesis of a Lys-Lys-linked cyclic leucine-enkephalin.

The side-chain-protected heptapeptide Phe-Leu-Lys(F3Ac)-Lys(F3Ac)-Tyr(Cac)-Gly-Gly is assembled by stepwise chain elongation on a polystyrene resin, using inter alia N-(9-xanthenyl)-amino acid N-carboxyanhydrides. After HBr cleavage from the resin, cyclization via carbodiimide/N-hydroxysuccinimide is carried out in a yield of 24%. Cleavage of the 9-carbazolylcarbonyl group by hydrazinolysis, and alkaline hydrolysis of the trifluoroacetyl groups, affords cyclo (-Tyr-Gly-Gly-Phe-Leu-Lys-Lys-) in an over-all yield of 12%. The structure of the peptides is confirmed by mass spectrometry.

Endorphins↗