Analysis of the interaction between the aspartic peptidase inhibitor SQAPI and aspartic peptidases using surface plasmon resonance

Aspartic peptidase inhibitors, which are themselves proteins, are strong inhibitors (small inhibition constants) of some aspartic peptidases but not others. However, there have been no studies of the kinetics of the interaction between a proteinaceous aspartic peptidase inhibitor and aspartic peptidases. This paper describes an analysis of rate constants for the interaction between recombinant squash aspartic peptidase inhibitor (rSQAPI) and a panel of aspartic peptidases that have a range of inhibition constants for SQAPI. Purified rSQAPI completely inhibits pepsin at a 1:1 molar ratio of pepsin to rSQAPI monomer (inhibition constant 1 nM). The interaction of pepsin with immobilized rSQAPI, at pH values between 3.0 and 6.0, was monitored using surface plasmon resonance. Binding of pepsin to rSQAPI was slow (association rate constants ca 10(4)M (-1)s(-1)), but rSQAPI was an effective pepsin inhibitor because dissociation of the rSQAPI-pepsin complex was much slower (dissociation rate constants ca 10(-4)s(-1)), especially at low pH values. Similar results were obtained with a His-tagged rSQAPI. Strong inhibition (inhibition constant 3 nM) of one isoform (rSap4) of the family of Candida albicans-secreted aspartic peptidases was, as with pepsin, characterized by slow binding of rSap4 and slower dissociation of the rSap4-inhibitor complex. In contrast, weaker inhibition of the Glomerella cingulata-secreted aspartic peptidase (inhibition constant 7 nM) and the C. albicans rSap1 and Sap2 isoenzymes (inhibition constants 25 and 400 nM, respectively) was, in each case, characterized by a larger dissociation rate constant.     

The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli.

The lacY from Escherichia coli strains 020 and AE43 have been cloned on plasmids which were designated p020-K358T and pAE43-D237N. These lacY mutants contain amino acid substitutions changing Lys-358 to Thr or Asp-237 to Asn, respectively. The charge neutralizing effect of each mutation is associated with a functional defect in melibiose transport which we exploited in order to isolate second site revertants to the melibiose-positive phenotype. Eleven melibiose-positive revertants of p020-K358T were isolated. All contained a second-site mutation converting Asp-237 to a neutral amino acid (8 to Asn, 1 to Gly, and 2 to Tyr). Twelve melibiose-positive revertants of pAE43-D237N were isolated. Two were second-site revertants converting Lys-358 to a neutrally Gln residue, while the remainder directly reverted Asn-237 to the wild-type Asp-237. We conclude that the functional intimate relationship between Asp-237 and Lys-358 suggests that these residues may be closely juxtaposed in three-dimensional space, possibly forming a 'charge-neutralizing' salt bridge.     

Kinetics of interaction between 3-hydroxyphthaloyl-beta-lactoglobulin and CD4 molecules.

Kinetics of 3-hydroxyphthaloyl-beta-lactoglobulin-CD4 interaction were evaluated using a biosensor instrument based on surface plasmon resonance. A very fast association (k(a)=2.4+/-0.3x10(6)M(-1)s(-1)) and slow dissociation (K(d)=2.3+/-0.14x10(-4)s(-1)) rate constants were observed indicating the high affinity of the complex. This result together with earlier data, suggest that "structure-specific" requirements must be met to endow acid anhydride modified lactoglobulin with the capacity for high affinity binding to CD4.     

Kinetics of the hemerythrin-oxygen interaction.

The kinetics of the reaction of Golfingia gouldii hemerythrin with O2 have been studied by stopped flow spectrophotometry. For the second order oxygenation process, k1 = 7.4 X 10(6) M-1 s-1, deltaH1++ = 8.2 kcal-mol-1 and deltaS1++ = +1 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The rate constant is unchanged when protein concentration is varied from 3 to 25 muM, the ionic strength is increased to 0.07 M, and the pH moved to 6.8. The deoxygenation of oxyhemerythrin is studied with stopped flow by scavenging liberated O2 with S2O4(2-). For the first order dissociation, k-1 = 51 s-1, deltaH-1++ = 20.6 kcal-mol-1 and deltaS-1++ = +19 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The value of k-1 is independent of [protein] = 50 to 200 muM, [S2O4(2-)] = 5 to 100 mM I = 0.015 to 0.30 M and pH 6.8 to 9.0. Using myoglobin instead of S2O4(2-) as scavenger gives similar results. Combination of activation parameters for the oxygenation and deoxygenation processes gives K1 = 1.5 X 10(5) M-1, deltaH = -12.4 kcal-mol-1, and deltaS = -18 e.u., values in good agreement with independent thermodynamic data. Perchlorate ion (0.05 M) enhances k-1 about 3-fold and hardly effects k1. There is no sign of other than a single reaction in either direction, and octameric hemerythrin apparently behaves kinetically as eight single units.     

亚甲蓝与多聚阴离子化合物结合反应的研究

对光谱探针亚甲蓝(MB)分别与硫酸化茯苓多糖(SP)、硫酸软骨素(CS)、透明质酸(HA)等多聚阴离子化合物相互作用的吸收光谱进行了比较研究,探讨了CS等多聚阴离子化合物与MB相互作用机理及其与MB结合的功能基团,计算了MB与SP、CS最大结合数分别为54和73。研究结果表明MB与多聚阴离子化合物的磺酸基发生明显的结合反应,与羧基不能发生明显的结合反应,多聚阴离子化合物与MB结合的功能基团是磺酸基。     

Kinetics and mechanism of the interaction between human serum albumin and monomeric haemin.

The interaction of human serum albumin with monomeric haemin has been investigated by detailed kinetic analysis in dimethyl sulphoxide/water (3:5, v/v). The results obtained under conditions of albumin saturation of haemin and under pseudo-single turnover conditions indicate that methaemalbumin is formed in a two-stage, single-intermediate process. The initial association between the haemin and human serum albumin is a chemically controlled process (k1 = 1.7 X 10(5) mol-1 . s-1 . dm3 at 24 degrees C); the variation of K1 with pH exhibited a well defined pK of 5.9. The overall equilibrium constant, calculated by using microscopic rate constants, is 1.1 (+/- 0.5) X 10(8) mol-1 at 24 degrees C. The data and conclusions are consistent with a general binding mechanism for albumin in which intermediate formation is followed by an entropy-controlled internalization of the ligand.     

Kinetics of reaction of anions with methemerythrin derivatives.

The kinetics of anation of methemerythrin over a wide range of pH and concentration of anions have been studied at 25 degrees C. The azide and thiocyanate ions have been most intensively investigated but experiments with fluoride and chloride are also reported. The replacement of anion in methemerythrin-anionic adducts by other anions has also been studied. Except for replacement of met-fluoride by azide, all replacements can be explained by a dissociative mechanism via the aquated species. Anations are second-order and an associative mechanism is preferred. The second-order rate constant decreases with increasing anion concentrations (from 20 muM to 20 mM). This is attributed to the effect of a secondary anion binding site. The behavior of octameric and monomeric forms of the protein toward thiocyanate is identical. A comparison of results with simple Fe(III) complexes and certain metalloproteins is made.     

Kinetics of redox interaction between substituted quinones and ascorbate under aerobic conditions.

One-electron reduction of quinones (Q) by ascorbate (AscH ); (1) AscH + Q --> Q*- + Asc*- + H+, followed by the oxidation of semiquinone (Q*-) by molecular oxygen; (2) Q*- + O2 --> Q + O2*-, results in the catalytic oxidation of ascorbate (with Q as a catalyst) and formation of active forms of oxygen. Along with enzymatic redox cycling of Q. this process may be related to Q cytotoxicity and underlie an antitumor activity of some Qs. In this work, the kinetics of oxygen consumption accompanied the interaction of ascorbate with 55 Qs including substituted 1,4- and 1,2-benzoquinones, naphthoquinones and other quinoid compounds were studied in 50 mM sodium phosphate buffer, pH 7.40, at 37 degrees C by using the Clark electrode technique. The capability of Q to catalyze ascorbate oxidation was characterized by the effective value of kEFF calculated from the initial rate of oxygen consumption (R(OX)) by the equation R(OX) = kEFF[Q][AscH-] as well as by a temporary change in R(OX). The correlation of kEFF with one-electron reduction potential, E(Q/Q*-), showed a sigma-like plot, the same for different kinds of Qs. Only the Qs which reduction potential E(Q/Q*-) ranged from nearly -250 to + 50 mV displayed a pronounced catalytic activity, kEFF increased with shifting E(Q/Q*-) to positive values. The following linear correlation between kEFF (in M (-1) s(-1)) and E(Q/Q*-) (in mV) might be suggested for these Qs: lg(kEFF)= 3.91 + 0.0143E(Q/Q*-). In contrast, Qs with E(Q/Q*-) < - 250 mV and E(Q/Q*-) > + 50 mV showed no measurable catalytic activity. The Qs studied displayed a wide variety in the kinetic regularities of oxygen consumption. When E(Q/Q*-) was more negative than - 100 mV, Q displayed a simple ('standard') kinetic behavior--R(OX) was proportional to [AscH-][Q] independently of concentration of individual reagents, [AscH-] and [Q]; R(OX) did not decrease with time if [AscH-] was held constant: Q recycling was almost reversible. Meanwhile, Qs with E(Q/Q*-) > - 100 mV demonstrated a dramatic deviation from the 'standard' behavior that was manifested by the fast decrease in R(OX) with time and non-linear dependence of even starting values of R(OX) on [Q] and [AscH-]. These deviations were caused basically by the participation of Q*- in side reactions different from (2). The above findings were confirmed by kinetic computer simulations. Some biological implications of Q-AscH- interaction were discussed.     

Density functional theory study on the interaction between keto-9H guanine and aspartic acid

A theoretical study was performed using density functional theory (DFT) to investigate hydrogen bonding interactions in signature complexes formed between keto-9H guanine (Gua) and aspartic acid (Asp) at neutral pH. Optimized geometries, binding energies and the theoretical IR spectra of guanine, aspartic acid and their corresponding complexes (Gua-Asp) were calculated using the B3LYP method and the 6-31+G(d) basis set. Stationary points found to be at local minima on the potential energy surface were verified by second derivative harmonic vibrational frequency calculations at the same level of theory. AIM theory was used to analyze the hydrogen bonding characteristics of these DNA base complex systems. Our results show that the binding motif for the most stable complex is strikingly similar to a Watson-Crick motif observed in the guanine-cytosine base pair. We have found a range of hydrogen bonding interactions between guanine and aspartic acid in the six complexes. This was further verified by theoretical IR spectra of ω(C-H---O-H) cm⁻¹ stretches for the Gua-Asp complexes. The electron density plot indicates strong hydrogen bonding as shown by the 2p _z dominant HOMO orbital character.     

Kinetics studies on the interaction between ouabain and (Na+,K+)-ATPase.

The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction.