Structure and mechanism of metallocarboxypeptidases

Metallocarboxpeptidases cleave C-terminal residues from peptide substrates and participate in a wide range of physiological processes, but they also contribute to human pathology. On the basis of structural information, we can distinguish between two groups of such metallopeptidases: cowrins and funnelins. Cowrins comprise protozoan, prokaryotic, and mammalian enzymes related to both neurolysin and angiotensin-converting enzyme and their catalytic domains contain 500-700 residues. They are ellipsoidal and traversed horizontally by a long, deep, narrow active-site cleft, in which the C-terminal residues are cut from oligopeptides and unstructured protein tails. The consensus cowrin structure contains a common core of 17 helices and a three-stranded beta-sheet, which participates in substrate binding. This protease family is characterized by a set of spatially conserved amino acids involved in catalysis, HEXXH+EXXS/G+H+Y/R+Y. Funnelins comprise structural relatives of the archetypal bovine carboxypeptidase A1 and feature mammalian, insect and bacterial proteins with strict carboxypeptidase activity. Their approximately 300-residue catalytic domains evince a consensus central eight-stranded beta-sheet flanked on either side by a total of eight helices. They also contain a characteristic set of conserved residues, HXXE+R+NR+H+Y+E, and their active-site clefts are rather shallow and lie at the bottom of a funnel-like cavity. Therefore, these enzymes act on a large variety of well-folded proteins. In both cowrins and funnelins, substrate hydrolysis follows a common general base/acid mechanism. A metal-bound solvent molecule ultimately performs the attack on the scissile peptide bond with the assistance of a strictly conserved glutamate residue.     

Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor.

The recombinant humanized antibody (rhuMAb) VEGF has a high affinity for vascular endothelial growth factor and is currently being evaluated in clinical trials as a cancer therapeutic. Under acidic pH and low ionic strength conditions, the antibody was predominantly present as monomer. Under physiological conditions, the appearance of significant amounts of a noncovalent, reversible dimer were observed by size-exclusion chromatography. The kinetics and thermodynamics of the reversible self-association for rhuMAb VEGF monomer were investigated as a function of pH, temperature, and ionic strength by size-exclusion chromatography using the concentration jump method. The rate constant for dimer formation ranged 23-112 M(-)(1) min(-)(1) under the conditions studied, values that are significantly lower than those reported in the literature for other proteins that self-associate. The rate constant for dissociation ranged 0.0039-0.021 min(-)(1). Gibbs' free energies, enthalpies, entropies, and activation energies were determined and revealed that dimer formation is optimal at pH 7.5-8.0, which may be reflective of charge shielding occurring near the pI of the protein. There was a negative change in entropy for dissociation (values from -18.1 to -12.8 cal/mol K). In the presence of D(2)O or 1 M NaCl, dimerization was enhanced. The results of the kinetic and thermodynamic analysis of this study indicate that rhuMAb VEGF dimerization occurs primarily through hydrophobic interactions.     

Kinetics and mechanism of dissociation of zinc ion from carbonic anhydrase.

The kinetics of dissociation of Zn2+ from the metalloenzyme carbonic anhydrase was measured over a range of pH, temperature, and acetate concentration. The rate of dissociation is extremely slow at neutral pH (t1/2 approximately 3) years, 4 degrees C), but increases in almost direct proportion to the hydrogen ion concentration and is enhanced in the presence of 1,10-phenanthroline or acetate. The thermodynamic stability of the zinc-apoenzyme complex was determined over a range of pH from rate data on binding and dissociation (stability constants 10(9)-10(11) M-1, 25 degrees C). The great stability of the complex and slow exchange of the apoenzyme ligand is attributed, at least in part, to the rigidity of the multidentate protein ligand.     

Kinetics of the formation and dissociation of actin filament branches mediated by Arp2/3 complex

The actin filament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from "mother" filaments growing near the plasma membrane. The nucleotide bound to the mother filaments (ATP, ADP and phosphate, or ADP) may influence the branch dynamics. To determine the effect of the nucleotide bound to the subunits of the mother filament on the formation and stability of branches, we compared the time courses of actin polymerization in bulk samples measured using the fluorescence of pyrene actin with observations of single filaments by total internal reflection fluorescence microscopy. Although the branch nucleation rate in bulk samples was nearly the same regardless of the nucleotide on the mother filaments, we observed fewer branches by microscopy on ADP-bound filaments than on ADP-P(i)-bound filaments. Observation of branches in the microscope depends on their binding to the slide. Since the probability that a branch binds to the slide is directly related to its lifetime, we used counts of branches to infer their rates of dissociation from mother filaments. We conclude that the nucleotide on the mother filament does not affect the initial branching event but that branches are an order of magnitude more stable on the sides of new ATP- or ADP-P(i) filaments than on ADP-actin filaments.     

Kinetics of the decamer - dimer dissociation of arginine decarboxylase.

Aurintricarboxylic acid inhibited replicative DNA synthesis in nucleotide-permeable mouse ascites sarcoma cells. DNA polymerase activity assayed with activated DNA template and DNA polymerase purified partially from sarcoma cells was also inhibited by aurintricarboxylic acid. The inhibition of DNA polymerase activity was probably due to the inhibitory interaction of aurintricarboxylic acid with DNA polymerase. The replicative DNA synthesis might be inhibited by aurintricarboxylic acid interacting with some essential protein component(s), such as DNA polymerase of the replication machinery.     

Kinetics of rouleau formation. II. Reversible reactions.

Red blood cells aggregate face-to-face to form long, cylindrical, straight chains and sometimes branched structures called rouleaux. Here we extend a kinetic model developed by R. W. Samsel and A. S. Perelson (1982, Biophys. J. 37:493-514) to include both the formation and dissociation of rouleaux. We examine thermodynamic constraints on the rate constants of the model imposed by the principle of detailed balance. Incorporation of reverse reactions allows us to compute mean sizes of rouleaux and straight chain segments within rouleaux, as functions of time and at equilibrium. Using the Flory - Stockmayer method from polymer chemistry, we obtain a closed-form solution for the size distribution of straight chain segments within rouleaux at any point in the evolution of the reaction. The predictions of our theory compare favorably with data collected by D. Kernick , A.W.L. Jay , S. Rowlands , and L. Skibo (1973, Can. J. Physiol. Pharmacol. 51:690-699) on the kinetics of rouleau formation. When rouleaux grow large, they may contain rings or loops and take on the appearance of a network. We demonstrate the importance of including the kinetics of ring closure in the development of realistic models of rouleaux formation.     

Autoxidation of alkoxylipids. III. Kinetics of peroxide formation

The influence of the type and position of various functional groups in saturated glycerol-derived alkoxylipids on the kinetics of peroxide formation is studied. The autoxidation of the glycerol-derived compounds is compared with that of some structural analogs. As a rule, ethers are oxidized much faster than ether-esters and esters. Free hydroxy groups exert an accelerating effect on the rate of autoxidation.     

Kinetics of formation of fibrin oligomers. I. Theory

The course of formation of fibrin oligomers is treated theoretically for the condition that self-assembly of fibrin monomers is rapid compared with the loss of A peptides by the enzymatic action of thrombin. The rate constant for removal of the second A peptide is taken to be larger than that for the first by an arbitrary factor q; the association of activated A sites with their complementary a sites is assumed to be random and independent of oligomer size. Two types of oligomers are considered: noncovalently bonded protofibrils formed by the staggered overlap of thrombin-activated monomers and covalently bonded linear oligomers formed by factor XIIIa-mediated end-to-end ligation of adjacent monomers within protofibrils. Oligomers of the first type, if ligated, are dissociated to oligomers of the second type by solubilization in SDS–urea. Theoretical curves are presented for x _w and x ′_w (weight-average degree of polymerization of staggered overlap and linear ligated oligomers, respectively) and for the weight fractions of monomer, dimer, and decamer of both ligated and unligated species as functions of y, the fraction of A peptide removed; and also for w_x and w′_x, the weight fractions of x-mer of the respective oligomer types, as a function of x at y = 0.5. With increasing q, the maximum w_x or w′_x that a low oligomer will reach during the reaction decreases and the size distribution is broadened toward larger oligomers. Comparison with experiment is made in a companion paper.     

Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation

The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.     

Kinetics of recA function in conjugational recombinant formation.

Summary Genetic recombination was studied in F^- strains of E. coli carrying a mutation (recA200) that confers a thermosensitive Rec^- phenotype. Recombination during Hfr matings at 35C was monitored by raising the temperature of incubation to 42C at various intervals so that only merozygotes that had completed those functions dependent on the activity of the recA gene product could form recombinant progeny. The results indicated that no more than 1–2% of the merozygotes present while mating was in progress were able to form recombinant colonies at 42C. Separation of mating pairs reduced the yield of recombinants obtained at 35C by 50 to 200-fold if plating on agar medium was delayed for 15–30min by continuing incubation in broth medium. recA200 merozygotes that were also recB21 sbcB15 proved relatively stable when plating was delayed in this manner, which suggested that Hfr DNA is prone to exonuclease inactivation in recA200 merozygotes after mating pairs have separated. Post-mating incubation in high salt medium or on agar plates promoted the recovery of recombinants at 35C. However, the majority of recA200 merozygotes did not acquire the ability to form recombinant colonies at 42C under these more stable conditions until mating pairs had been separated and incubation continued at 35C for 40–60 min. It was concluded that recA200 strains are partially defective for recombination even at low temperature but that terminating mating promotes the recovery of recombinants. A mechanism involving the stimulation of RecA activity by mating pair separation is postulated to account for the efficient recovery of recombinants from HfrxF^- recA200 crosses at 35C.     

Kinetics of dissociation of parvalbumin complexes with calcium and magnesium ions

Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit.     

Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium measurements under the same conditions. These results are consistent with sequential dissociation of calcium from a narrow binding pocket inside which a single calcium ion can move fairly easily. Escape of calcium might be controlled by a structural compartment acting as a gate.     

Spin-labeled gramicidin a: channel formation and dissociation

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.     

Kinetics of cadmium and terbium dissociation from calmodulin and its tryptic fragments

The kinetics of cadmium and terbium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by stopped-flow fluorescence methods, using the calcium indicator Quin 2. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, have clearly identified cadmium binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. Thus the site preference of cadmium is the same as that of calcium. For terbium, however, sites I and II are the high-affinity sites and sites III and IV are the low-affinity sites. Thus, the site preference or terbium is not the same as that of calcium and cadmium. In contrast to previous studies with calcium, we observe two kinetic processes for dissociation from sites III and IV for experiments with both cadmium and terbium. Possible models for the binding of metal ions are discussed.     

Progress in metallocarboxypeptidases and their small molecular weight inhibitors

In what corresponds to a life span, metallocarboxypeptidases (MCPs) have jumped from being mere contaminants in animal pancreas powders (in depression year 1929) to be key players in cellular and molecular processes (in yet-another-depression years 2009–2010). MCPs are unique zinc-dependent enzymes that catalyze the breakdown of the amide bond at the C-terminus of peptide and protein substrates and participate in the recovery of dietary amino acids, tissue organogenesis, neurohormone and cytokine maturation and other important physiological processes. More than 26 genes code for MCPs in the human genome, many of them still waiting to be fully understood in terms of physiological function. A variety of MCPs have been linked to diseases in man: acute pancreatitis and pancreas cancer, type 2 diabetes, Alzheimer’s Disease, various types of cancer, and fibrinolysis and inflammation. Many of these discoveries have been made possible thanks to recent advances, as exemplified by plasma carboxypeptidases N and B, known for fifty and twenty years, respectively, which have had their structures released only very recently. Plasma carboxypeptidase B is a biological target for therapy because of its involvement in the coagulation/fibrinolysis processes. Besides, the widespread use of carboxypeptidase A as a benchmark metalloprotease since the early days of Biochemistry has allowed the identification and design of an increasingly vast repertory of small molecular weight inhibitors. With these two examples we wish to emphasize that MCPs have become part of the drug discovery portfolio of pharmaceutical companies and academic research laboratories. This paper will review key developments in the discovery and design of MCP small molecular weight inhibitors, with an emphasis on the discovery of chemically diverse entities. Although encouraging advances have been achieved in the last few years, the specificity and oral bioavailability of the new chemotherapeutic agents seem to pose a challenge to medicinal chemists.