Use of binding energy in catalysis analyzed by mutagenesis of the tyrosyl-tRNA synthetase

The utilization of enzyme-substrate binding energy in catalysis has been investigated by experiments on mutant tyrosyl-tRNA synthetases that have been generated by site-directed mutagenesis. The mutants are poorer enzymes because they lack side chains that form hydrogen bonds with ATP and tyrosine during stages of the reaction. The hydrogen bonds are not directly involved in the chemical processes but are at some distance from the seat of reaction. The free energy profiles for the formation of enzyme-bound tyrosyl adenylate and the equilibria between the substrates and products were determined from a combination of pre-steady-state kinetics and equilibrium binding methods. By comparison of the profile of each mutant with wild-type enzyme, a picture is built up of how the course of reaction is affected by the influence of each side chain on the energies of the complexes of the enzyme with substrates, transition states, and intermediates (tyrosyl adenylate). As the activation reaction proceeds, the apparent binding energies of certain side chains with the tyrosine and nucleotide moieties increase, being weakest in the enzyme-substrate complex, stronger in the transition state, and strongest in the enzyme-intermediate complex. Most marked is the interaction of Cys-35 with the 3'-hydroxyl of the ribose. Removal of the side chain of Cys-35 leads to no change in the dissociation constant of ATP but causes a 10-fold lowering of the catalytic rate constant. It contributes no net apparent binding energy in the E X Tyr X ATP complex and stabilizes the transition state by 1.2 kcal/mol and the E X Tyr-AMP complex by 1.6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

Active site mechanics of liver microsomal cytochrome P-450

For a set of 10 para-substituted toluene derivatives, three enzymatic constants were determined describing their interaction with purified rabbit liver microsomal P-450LM2. The three constants were the catalytic rate constant (Kcat) for hydroxylation, the apparent dissociation constant (Kd) for the enzyme-substrate complex, and the interaction energy (delta Gint) between the substrate-binding and spin-state equilibria. The para-substituents of the toluene substrates were: hydrogen, fluoro, bromo, chloro, iodo, nitro, methyl, cyano, isopropyl, and t-butyl. Linear free energy correlations were sought between the enzymatic constants and several physical constants of the individual substrate molecules. These correlations would be useful both for empirical prediction purposes and for insight into active site chemistry and mechanics. Catalytic rates were correlated by a linear combination of the Hansch pi hydrophobic constant and the Hammett sigma value. A deuterium isotope effect (DV) of 2.6 for d8-toluene compared to d0-toluene confirmed that hydrogen abstraction was partially rate-limiting with this series of substrates. Apparent dissociation constants were predicted by a linear combination of the molar volume and pi, while the spin-state interaction energies were best predicted by a linear combination of the Hansch pi hydrophobic constant and the reciprocal of the dielectric constant.     

Evidence of essential arginyl residues in chicken liver mevalonate-5-pyrophosphate decarboxylase

Chicken liver mevalonate-5-pyrophosphate decarboxylase (ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33.) is inactivated by phenylglyoxal in triethanolamine buffer at pH 8.15. The reaction follows pseudo-first-order kinetics with a second-order rate constant of 108 M-1 min-1. Appropriate treatment of the kinetic data for the inactivation reaction indicates that the reaction of a single phenylglyoxal molecule per active unit of the enzyme is enough to completely inactivate the protein. The partially inactivated enzyme shows unaltered Km but decreased V as compared to native mevalonate-5-pyrophosphate decarboxylase. The dissociation constants for the enzyme-substrate complexes were estimated from inactivation reactions at different concentrations of substrates. From the data it is concluded that the modified amino acid is important for the binding of both substrates.     

Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.     

Duration of courtship effort as a costly signal

We consider a male and a female in a courtship encounter over continuous time. Both parties pay participation costs per unit time. The game ends when either one or other of the parties quits or the female accepts the male as a mate. We assume that there is a binary variable which determines whether the male is a “good” or “bad” type from the female's point of view, according to either his condition or his willingness to care for the young after mating. This variable is not directly observable by the female, but has fitness consequences for her: she gets a positive fitness payoff from mating with a “good” male but a negative fitness payoff from mating with a “bad” male. We assume also that a “good” male has a higher ratio of fitness benefit from mating to fitness cost per unit time of courtship than a “bad” male. We show that, under suitable assumptions, there are evolutionarily stable equilibrium behaviours in which time-extended courtship takes place. A “good” male is willing to court for longer than a “bad” male; in this way the duration of a male's courtship signals his type, and acts as a costly handicap. By not being willing to mate immediately the female achieves a degree of screening because the posterior probability that the male is “good”, conditional on his not having quit the game, increases with the duration of courtship.     

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the “more reactive” and “less reactive” conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.     

The mechanism of soluble peptidoglycan hydrolysis by an autolytic muramidase. A processive exodisaccharidase

The action of purified N-acetylmuramoylhydrolase (muramidase, EC 3.2.1.17) of Streptococcus faecium ATCC 9790 on linear, uncross-linked, soluble, peptidoglycan chains produced by the same organism in the presence of benzylpenicillin was characterized as a processive exodisaccharidase. Specific labels, one [( 14C]Gal) added to the nonreducing ends of chains, and the other (3H from [3H]NaBH4) incorporated into the reducing ends of the chains, were used to establish that an enzyme molecule binds at the nonreducing terminus and sequentially hydrolyzes the glycosidic bonds, releasing disaccharide-peptide units. An enzyme molecule remains bond to a chain, and is not released at a detectable rate, until hydrolysis of that chain is complete. Reaction rates increased with the length of the polymer chain to give a maximum of 91 bonds cleaved/min/enzyme molecule for hydrolysis of a continuous polymeric substrate. The relationship between hydrolytic rate and glycan chain length is consistent with hydrolysis of bonds within the chain followed by slow release of enzyme from the distal, reducing terminus. This mechanism was experimentally confirmed by analysis of product formation during hydrolysis with stoichiometric mixtures of enzyme and soluble peptidoglycan chains. Kinetic analyses showed an apparent Km of 0.17 microM for the enzyme, independent of substrate polymer length. The dissociation constant for the initial enzyme-substrate complex was calculated to be 1.5 nM. Kinetic analyses are consistent with one catalytic site per enzyme molecule. The Kcat/Km value of 9 X 10(6) M-1 S-1 is near the limit imposed by diffusion for the initial hydrolytic events when long chains are hydrolyzed. The kinetic and physical properties of this muramidase are highly consistent with its location outside of the cellular permeability barrier and its ability to remain with and hydrolyze appropriate bonds in the cell wall in such an environment.     

The distribution of the coalescence time and the number of pairwise nucleotide differences in a model of population divergence or speciation with an initial period of gene flow

This paper is concerned with a model of “isolation with an initial period of migration”, where a panmictic ancestral population split into n descendant populations which exchanged migrants symmetrically at a constant rate for a period of time and subsequently became completely isolated. In the limit as the population split occurred an infinitely long time ago, the model becomes an “isolation after migration” model, describing completely isolated descendant populations which arose from a subdivided ancestral population. The probability density function of the coalescence time of a pair of genes and the probability distribution of the number of pairwise nucleotide differences are derived for both models. Whilst these are theoretical results of interest in their own right, they also give an exact analytical expression for the likelihood, for data consisting of the numbers of nucleotide differences between pairs of DNA sequences where each pair is at a different, independent locus. The behaviour of the distribution of the number of pairwise nucleotide differences under these models is illustrated and compared to the corresponding distributions under the “isolation with migration” and “complete isolation” models. It is shown that the distribution of the number of nucleotide differences between a pair of DNA sequences from different descendant populations in the model of “isolation with an initial period of migration” can be quite different from that under the “isolation with migration model”, even if the average migration rate over time (and hence the total number of migrants) is the same in both scenarios. It is also illustrated how the results can be extended to other demographic scenarios that can be described by a combination of isolated panmictic populations and “symmetric island” models.     

A study of the complex-formation of phenylalanyl-tRNA-synthetase from Escherichia coli using the tRNA-Phe method of small-angle x-ray scattering

The method of small-angle X-ray scattering was employed to analyse the equilibrium enzyme-substrate complexes in solution. A new approach of analysis of the experimental data was developed. This type of analysis provides the determination of dissociation constants and structural parameters of enzyme-substrate complexes. The radius of gyration (Rg) and dimensions of half-axis of the equivalent prolonged ellipsoid (a, b) of E. coliphenylalanyl-tRNA synthetase and its complexes with one or two tRNA(Phe) molecules have been determined. The values of these parameters speak in favour of structural rearrangements due to the interaction of the enzyme with tRNA(Phe). The thermodynamic characteristics of phenylalanyl-tRNA synthetase complexes with tRNA(Phe) testify to the negative cooperativity in binding of two tRNA molecules with the enzyme.     

The crystal and molecular structures of a cathepsin K:chondroitin sulfate complex

Cathepsin K is the major collagenolytic enzyme produced by bone-resorbing osteoclasts. We showed earlier that the unique triple-helical collagen-degrading activity of cathepsin K depends on the formation of complexes with bone-or cartilage-resident glycosaminoglycans, such as chondroitin 4-sulfate (C4-S). Here, we describe the crystal structure of a 1:n complex of cathepsin K:C4-S inhibited by E64 at a resolution of 1.8 Å. The overall structure reveals an unusual “beads-on-a-string”-like organization. Multiple cathepsin K molecules bind specifically to a single cosine curve-shaped strand of C4-S with each cathepsin K molecule interacting with three disaccharide residues of C4-S. One of the more important sets of interactions comes from a single turn of helix close to the N terminus of the proteinase containing a basic amino acid triplet (Arg8-Lys9-Lys10) that forms multiple hydrogen bonds either to the caboxylate or to the 4-sulfate groups of C4-S. Altogether, the binding sites with C4-S are located in the R-domain of cathepsin K and are distant from its active site. This explains why the general proteolytic activity of cathepsin K is not affected by the binding of chondroitin sulfate. Biochemical analyses of cathepsin K and C4-S mixtures support the presence of a 1:n complex in solution; a dissociation constant, K_d, of about 10 nM was determined for the interaction between cathepsin K and C4-S.     

Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases

The equilibrium constant for the formation of tyrosyl adenylate and pyrophosphate from ATP and tyrosine in solution has been measured by applying the Haldane relationship to wild-type and three mutant tyrosyl-tRNA synthetases from Bacillus stearothermophilus. The formation constant (=[Tyr-AMP] [PPi]/[ATP] [Tyr]) at pH 7.78, 25 degrees C, and 10 mM MgCl2 is (3.5 +/- 0.5) X 10(-7). This corresponds to a free energy of hydrolysis of tyrosyl adenylate at pH 7.0 and 25 degrees C of -16.7 kcal mol-1. All necessary rate constants had been determined previously for the calculations apart from the dissociation constant of tyrosyl adenylate from its enzyme-bound complex. This was measured by taking advantage of the 100-fold difference in hydrolysis rates of the tyrosyl adenylate when sequestered by the enzyme and when free in solution. These are technically difficult measurements because the dissociation constants are so low and the complexes unstable. The task was simplified by using mutants prepared by site-directed mutagenesis. These were designed to have different rate and equilibrium constants for dissociation of tyrosyl adenylate from the enzyme-bound complexes. The dissociation constants were in the range (3.5-38) X 10(-12) M, with that for wild type at 13 X 10(-12) M. The four enzymes all gave consistent data for the formation constant of tyrosyl adenylate in solution. This not only improves the reliability of the measurement but also provides confirmation of the reliability of the measured kinetic constants for the series of enzymes.     

The effect of mercury accumulation on the intestinal glycosidase activity in perch <Emphasis Type="Italic">Perca fluviatilis</Emphasis> L. from aquatic bodies of European Russia with different pH

The intestinal glycosidase activities and kinetic characteristics of carbohydrate hydrolysis change in different ways with mercury accumulation in the muscles of perch from aquatic bodies of European Russia. Amylolytic activity decreases in fish from aquatic bodies with a neutral pH with a growth in Hg content (0.05–0.30 mg per 1 kg fresh weight), whereas sucrase increases. A decrease in the Michaelis constant for carbohydrate hydrolysis reflects an adaptive increase in enzyme-substrate affinity. In fish from acid lakes, the muscle Hg accumulation amounts to 0.18–0.86 mg per 1 kg fresh weight. With a growth in Hg content, the glycosidase activities and enzyme-substrate affinity decrease in most cases, having a negative effect on the assimilation rate of the carbohydrate components of food.     

Complexes of bivalent cations with neryl and geranyl pyrophosphate: Their role in terpene biosynthesis

Kinetic analysis of the nonenzymic solvolysis of neryl and geranyl pyrophosphate (NPP and GPP, respectively) showed that the dissociation constants of the bis-metallic complexes with Mg²⁺ and Mn²⁺ were larger for NPP than for GPP by approximately one order of magnitude. Rate constants for reaction of the bis-metallic complexes were larger for NPP than for GPP. Qualitatively similar behavior was observed with complexes of Co²⁺. Extents of elimination and cyclization were increased by metal ions. Carbocyclase-catalyzed formation of cyclic monoterpene hydrocarbons in the presence of Mg²⁺ involved bis-metallic complexes as the “true” substrates.     

Detection and characterization of an additional site for binding of substrate and its analogs by inorganic pyrophosphatase

Phosphate, pyrophosphate, imidodiphosphate, EDTA and tripolyphosphate increase the rate constant for dissociation of the inorganic pyrophosphatase-substrate intermediate formed after cessation of the reaction by fluoride. The effect is enhanced in the given order 19-fold, the dependence of this effect on ligand concentration being hyperbolic. The values of the dissociation constants of the enzyme-ligand complexes lie within the concentration range of 0.16-1.0 mM. At high concentrations of Na2+ added simultaneously with the ligands this effect is decreased. The value of tau 1/2 for Pi binding to the enzyme-substrate compound is 0.15 min. The data obtained suggest that pyrophosphatase contains an anion ligand binding site, differing from that of the active one. This site does not affect the hydrolytic function of pyrophosphatase, as can be evidenced from the fact that Pi (9.5 mM) does not change the rate of enzymatic cleavage of PPi.     

Yeast orotidine-5'-phosphate decarboxylase: steady-state and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation

The catalytically active form of monofunctional yeast orotidine-5'-phosphate decarboxylase was a dimer (E(2)). The dimer equilibrium dissociation constant was 0.25 microM in 0.01 M MOPS Na(+) at pH 7.2. The bimolecular rate constant for dimer formation was 1.56 microM(-1) s(-1). The dimeric form of the enzyme was stabilized by NaCl such that the enzyme was E(2) in 100 mM NaCl at all concentrations of enzyme tested. The kinetics of binding of OMP to E(2) was governed by two ionizations (pK(1) = 6.1 and pK(2) = 7.7). From studies with substrate analogues, the higher pK was assigned to a group on the enzyme that interacted with the pyrimidinyl moiety. The value of the lower pK was dependent on the substrate analogue, which suggested that it was not exclusively the result of ionization of the phosphoryl moiety. During the decarboxylation of OMP, the fluorescence of E(2) was quenched over 20%. The enzymatic species with reduced fluorescence was a catalytically competent intermediate that had kinetic properties consistent with it being the initial enzyme-substrate complex. The stoichiometry for binding of OMP to E(2) was one OMP per enzyme monomer. The value of the first-order rate constant for conversion of the enzyme-substrate complex to free enzyme (36 s(-1)) calculated from a single turnover experiment ([E] > [S]) was slightly greater than the value of k(cat), 20 s(-1) (corrected for stoichiometry), calculated from steady-state data. In the single turnover experiments, the enzyme was E(2)*S, whereas in the steady-state turnover the experiment enzyme was E(2)*S(2). The similarity of these values suggested that the subunits were catalytically independent such that E(2)*S(2) could be treated as E*S and that conversion of the enzyme-substrate complex to E was k(cat). Kinetic data for the approach to the steady-state with OMP and E(2) yield a bimolecular association rate complex of 62 microM(-1) s(-1)and a dissociation rate constant for E*S of 60 s(-1). The commitment to catalysis was 0.25. By monitoring the effect of carbonic anhydrase on [H(+)] changes during a single turnover experiment, the initial product of the decarboxylation reaction was shown to be CO(2) not HCO(3-). UMP was released from the enzyme concomitantly with CO(2) during the conversion of E*S to E. Furthermore, the enzyme removed an enzyme equivalent of H(+) from solvent during this step of the reaction. The bimolecular rate constants for association of 6-AzaUMP and 8-AzaXMP, substrate analogues with markedly different nucleobases, had association rate constants of 112 and 130 microM(-1) s(-1), respectively. These results suggested that the nucleobase did not contribute significantly to the success of formation of the initial enzyme-substrate complex.     

Physical and chemical characterization of soil and poultry litter humic and fulvic metal complexes

Summary Humic and fulvic-zinc complexes obtained from soil and poultry litter were characterized by I.R. spectroscopy, determination of stability constant and the free energy change associated with their formation. Infrared spectroscopy confirmed that both electrovalent, coordinate-covalent bonds of Zn²⁺ with the carboxylate, phenolic hydroxyl and amine groups lead to the formation of their stable complexes. This is evident from the changes in absorption bands at 1700 cm^–1, 1725 cm^–1, 1625 cm^–1 and 1400 cm^–1 of their infrared spectra.The stability constants of complexes are pH-dependent. Interaction of Zn with humic and fulvic acid involved the formation of mononuclear complexes. The values of stability constants of these complexes are lower than those reported earlier.The calculation of the free-energy change associated with salts and complex formation indicates the spontaneity of both reactions, although a higher probability of complex reaction than that of salt formation is evident. The implications of the complexation of metal ions by these naturally occurring polydisperse plyaanions in regulating the movement of metal ions from the ambient soil matrix to plant roots and biological system in terresterial and aquatic environments are indicated.Journal paper no. 2, Department of Soil Science and Agricultural Chemistry, Rajendra Agricultural University, Tirhut College of Agriculture, Dholi, Muzaffarpur, Bihar, India.     

Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities.

Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 and several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), and d(CGCGAATTCGCG) all have dissociation constants in the range (1-3) X 10(-9) M-1. Such complexes on calf thymus DNA occur with a frequency of about 1 binding site per 100 base pairs, and evidence is presented indicating a spectrum of sequence-dependent affinities with dissociation constants extending into the micromolar range. In addition to these sequence-specific binding sites on the DNA, the continuous-variation method of Job reveals distinct stoichiometries of dye-poly[d(A-T)] complexes corresponding to 1, 2, 3, 4, and 6 dyes per 5 A-T base pairs and even up to 1 and 2 (and possibly more) dyes per backbone phosphate. Models are suggested to account for these stoichiometries. With poly[d(G-C)] the stoichiometries are 1-2 dyes per 5 G-C pairs in addition to 1 and 2 dyes per backbone phosphate. Thermodynamic parameters for formation of the tightest binding complex between Hoechst 33258 and poly[d(A-T)] or d-(CCGGAATTCCGG) are determined. Hoechst 33258 binding to calf thymus DNA, chicken erythrocyte DNA, and poly[d(A-T)] exhibits an ionic strength dependence similar to that expected for a singly-charged positive ion. This ionic strength dependence remains unchanged in the presence of 25% ethanol, which decreases the affinity by 2 orders of magnitude. In addition, due to its strong binding, Hoechst 33258 easily displaces several intercalators from DNA.     

Kinetics of the action of papain on fluorescent peptide substrates.

Kinetic measurements have been performed on the action of papain on mansyl-Gly-Val-Glu-Leu-Gly and on mansyl-Gly-Gly-Val-Glu-Leu-Gly, both of which are cleaved solely at the Glu-Leu bond under the conditions of our experiments. Stopped-flow experiments have shown that, under conditions of enzyme excess, the enhancement of the fluorescence of the mansyl group upon association of each of the oligopeptide substrates with papain is a biphasic process. A very rapid initial increase in fluorescence is followed by a slower first-order fluorescence enhancement. The observed rate constant for the latter process is greater with the mansyl pentapeptide than with the mansyl hexapeptide. A similar biphasic fluorescence change is seen upon the interaction of the mansyl peptides with mercuripapain, but the second step is much slower than in the case of the active enzyme. The rate of the second step in the association of active papain with the mansyl paptides shows saturation with increasing enzyme concentration, supporting the view that an initial enzyme-substrate complex (ES) is converted in a first-order process to the complex (ES) that undergoes cleavage to form products. The hydrolysis of the Glu-Leu bond is associated with a first-order decrease in fluorescence, as a consequence of the formation of the mansyl peptide product, which is bound less strongly than the substrate. The rate constant for this process is about 140 times greater with the mansyl hexapeptide than with the mansyl pentapeptide, thus giving further indication of the importance of secondary enzyme-substrate interactions in the efficiency of papain catalysis. For each of the two mansyl peptides, the values of the rate constants and the apparent Michaelis constants associated with the cleavage of the Glu-Leu bond, as determined by stopped-flow measurements under conditions of enzyme excess, were the same, within the precision of the data, as those estimated from experiments under conditions of substrate excess, where the formation of Leu-Gly was determined by means of the fluorescamine reaction. This indicates that, with these substrates, the rate-limiting step in the overall catalytic process is associated with the breakdown of ES. Estimates are given of the dissociation constant of ES and of the rate constants in the interconversion of ES and ES.     

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation.