Analysis of phi and chi 1 torsion angles for hen lysozyme in solution from 1H NMR spin-spin coupling constants

Three-bond 3JHN alpha coupling constants have been determined for 106 residues and 3J alpha beta coupling constants have been measured for 57 residues of the 129-residue protein hen egg white lysozyme. These NMR data have been compared with torsion angles defined in the tetragonal and the triclinic crystal forms of the protein. For most residues the measured 3JHN alpha values were consistent with the phi torsion angles found in both crystal forms; the RMS difference between the coupling constants calculated by using the tetragonal crystal structure phi angles and the experimental 3JHN alpha values is 0.88 Hz. Thus there appears to be no significant averaging of the phi torsion angle either in the interior or at the surface of the protein. For 41 of the residues where 3J alpha beta coupling constants have been determined, the values are consistent with a single staggered conformation about the chi 1 torsion angle and there is complete agreement between the NMR data in solution and the torsion angles defined in the crystalline state. In contrast, for the other 16 residues where 3J alpha beta coupling constant values have been measured, the data indicate extensive motional averaging about the chi 1 torsion angle. These residues occur largely on the surface of the protein and examination of the crystal structures shows that many of these residues adopt a different conformation in the triclinic and tetragonal crystal forms and have high crystallographic temperature factors. It appears, however, that in solution conformational flexibility of the side chains of surface residues is significantly more pronounced than in individual crystal structures.     

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.     

Kinetic properties of crystalline enzymes. Carboxypeptidase A.

Spectrochemical probes have demonstrated that the conformations of carboxypeptidase A differ in solution and in the crystalline state. Detailed kinetic studies of carboxypeptidase A crystals and solutions now show that the physical state of the enzyme is also a critical parameter that affects this enzyme's function. Thus, for all substrates examined, crystallization of the enzyme markedly reduces catalytic efficiency, kcat, from 20- to 1000-fold. In addition, substrate inhibition, apparent in solution for some di- and depsipeptides, is abolished with crystals, while longer substrates with normal kinetics in solution may exhibit activation with the crystals. The physical state of the enzyme also affects the mode of action of known modifiers of peptidase activity of the enzyme. In solution, addition of benzoylglycine or cinnamic acid markedly increases the rate of hydrolysis of CbzGly-Phe, but, with the crystalline enzyme, their addition hardly alters the activity. This is in accord with the weakening or absence of inhibitory enzyme-substrate binding modes. Kinetic studies on crystals were carried out over a range of enzyme concentrations, substrate concentrations, and crystal sizes, and in all instances the results are in good agreement with the theory developed by Katchalski for enzymes insolubilized by other means. Importantly, these kinetic parameters are determined under conditions which obviate artifacts due to diffusion limitation of substrates or products. The differences in the kinetic behavior of carboxypeptidase crystals, on the one hand, and of their solutions, on the other hand, bear importantly on efforts to interpret the function of the enzyme in structural terms. Hypothetical modes of substrate-enzyme interaction, generated by superimposing substrate models on the crystal structure of carboxypeptidase to stimulate kinetics in solution, have failed to detect all of these changes which affect inhibitory or activating binding modes.     

Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations.

Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.     

Kinetic investigation of the alpha-chymotrypsin-catalyzed hydrolysis of peptide-ester substrates. The relationship between the structure of the peptide moiety and reactivity.

A number of peptide-ester substrates of the general structure Ac-Lxn-...-Lx2-Lx1-OMe have been synthesized and their alpha-chymotrypsin-catalyzed hydrolysis studied. The kinetic analysis involved varying the concentration of substrate and methanol product, and measuring rates along the entire progression curve. For the dipeptide esters Ac-Lx2-Lx1-OMe and the amino-acid derivatives Ac-Lx1-OMe the following constants could be determined: the dissociation constant of the enzyme-substrate complex, KEA, both rate constants of the acylation step, k23 and k32, and the forward rate constant of the deacylation step, k31. For the tripeptide ester Ac-Ala-Ala-Tyr-OMe it appears that the rate constant for the dissociation of the enzyme-substrate complex, k21, is smaller than the rate constant for acylation, k23. Thus, for this substrate only the association and dissociation rate constants k12 and k21 could be determined and the values of k23, k32 and k31 only indirectly estimated. The influence of structural changes in the peptide moiety of the substrates on reactivity has been established by comparing the rate constants of appropriate pairs of substrates. It was found that the substrate reactivity, as measured by k23/KEA, increase with the number and strength of the secondary interactions in a manner consistent with the binding scheme which has been proposed on the basis of crystallographic studies. The effect of a particular interaction on k23 and on KEA is dependent on the nature of the other interactions. However, the effect of k23/KEA appears to be independent of the presence of the other interactions and therefore characteristic of that particular interaction. The results for these substrates are compared with those found previously for a series of peptide substrates of the structure Ac-Lxn-... Lx2-...-Lx1-Gly-NH2 which have the same acyl moiety as the peptide esters studied in this work.     

Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.     

Interactions of substrates at the surface of P450s can greatly enhance substrate potency

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.     

Modeling and synthesis of novel tight-binding inhibitors of cytochrome P450 2C9

Cytochrome P450 2C9 (2C9) is one of the three major drug metabolizing cytochrome P450 enzymes in human liver. Although the crystal structure of 2C9 has been solved, the important physicochemical properties of substrate-enzyme interactions remain difficult to be determined. This is due in part to the conformational flexibility of mammalian P450 enzymes. Therefore, probing the active-site with high-affinity substrates is important in further understanding substrate-enzyme interactions. Three-dimensional quantitative structure-activity relationships (3D-QSAR) and docking experiments have been shown to be useful tools in correlating biological activity with structure. In particular we have previously reported that the very tight-binding inhibitor benzbromarone can provide important information about the active-site of 2C9. In this study we report the binding affinities and potential substrate-enzyme interactions of 4H-chromen-4-one analogs, which are structurally similar to benzbromarone. The chromenone structures are synthetically accessible inhibitors and give inhibition constants as low as 4.2 nM, comparable with the very tightest-binding inhibitors of 2C9. Adding these compounds to our previous 2C9 libraries for CoMFA models reinforces the important electrostatic and hydrophobic features of substrate binding. These compounds have also been docked in the 2C9 crystal structure and the results indicate that Arg 108 plays significant roles in the binding of chromenone substrates.     

Identification of internal autoproteolytic cleavage sites within the prosegments of recombinant procathepsin B and procathepsin S. Contribution of a plausible unimolecular autoproteolytic event for the processing of zymogens belonging to the papain family

The steps involved in the maturation of proenzymes belonging to the papain family of cysteine proteases have been difficult to characterize. Intermolecular processing at or near the pro/mature junction, due either to the catalytic activity of active enzyme or to exogeneous proteases, has been well documented for this family of proenzymes. In addition, kinetic studies are suggestive of a slow unimolecular mechanism of autoactivation which is independent of proenzyme concentration. However, inspection of the recently determined x-ray crystal structures does not support this evidence. This is due primarily to the extensive distances between the catalytic thiolate-imidazolium ion pair and the putative site of proteolysis near the pro/mature junction required to form mature protein. Furthermore, the prosegments for this family of precursors have been shown to bind through the substrate binding clefts in a direction opposite to that expected for natural substrates. We report, using cystatin C- and N-terminal sequencing, the identification of autoproteolytic intermediates of processing in vitro for purified recombinant procathepsin B and procathepsin S. Inspection of the x-ray crystal structures reported to date indicates that these reactions occur within a segment of the proregion which binds through the substrate binding clefts of the enzymes, thus suggesting that these reactions are occurring as unimolecular processes.     

Solution structure of porcine pancreatic phospholipase A2.

The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.     

Characterization of antigen-binding receptors in vitro. II. Antigen-binding capacity and affinity of lymphoid receptors after immunization.

We examined the kinetics and affinity of antigen binding in lymphoid populations in mice after immunization. There is increased binding capacity in lymphoid cells from animals that have undergone primary immunization. This increase would seem to be related to increased numbers of antigen-binding cells (rosette-forming cells). The serum antibody titers rise after the increasing binding capacity and numbers of BSA rosette-forming cells have increased. There is an increased amount of antigen bound per antigen-binding cell at certain times after immunization with two peaks in this capacity being demonstrable--one occurring at 4 days after immunization and the second occurring approximately 12 days after immunization and persisting for prolonged periods after that. With time, after immunization two separable peaks of increased antigen-binding cells become apparent, one very early (before Day 4) and one later (after Day 20 to 30). The affinity constants for antigen-binding cells have been measured and found to be high, and to increase with time after immunization. It appears that the heterogeneity of the affinity constants for antigen-binding cells is high early in immunity and becomes more homogeneous with time after immunization.     

A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction.

We have investigated the chemical basis for a previously observed 7.8 A conformational change in the hammerhead ribozyme that positions the substrate for in-line attack. We have found that the conformational change can only be observed at or above pH 8.5 (in the presence of Co(2+)) and requires the presence of an ionizable 2'-OH at the cleavage site, and note that this observed apparent pK(a) of 8.5 for the conformational change is within experimental error (+/-0.5) of the previously reported apparent kinetic pK(a) of 8.5 for the hammerhead ribozyme in the presence of Co(2+). We have solved two crystal structures of hammerhead ribozymes having 2'-OCH(3) or 2'-F substitutions at the cleavage site and have found that these will not undergo a conformational change equivalent to that observed for the hammerhead ribozyme having an unmodified attacking nucleophile under otherwise identical conditions. We have also characterized the kinetics of cleavage in the crystal. In addition to verifying that the particular sequence of RNA that we crystallized cleaves faster in the crystal than in solution, we also find that the extent of cleavage in the crystal is complete, unlike in solution where this and most other hammerhead ribozyme substrates are cleaved only to about 70 % completion. The initial cleavage rate in the crystal obeys the expected log-linear relation between cleavage-rate and pH with a slope of 0.7, as has been observed for other hammerhead ribozyme sequences in solution, indicating that in both the crystal and in solution the pH-dependent step is rate-limiting. However, the cleavage rate in the crystal is biphasic, with the most dramatic distinction between initial (slower) and final (faster) phases appearing at pH 6.0. The initial phase corresponds to the pH-dependent cleavage rate observed in solution, but the second, faster phase is roughly pH-independent and closely parallels the cleavage rate observed at pH 8 (0.4/minute). This result is particularly remarkable because it entails that the rapidly cleaving phase at pH 6 is comparable to the cleavage rate for the fastest cleaving hammerhead ribozymes at pH 6. Based upon these observations, we conclude that the pH-dependent conformational change is the rate-determining step under standard conditions for the hammerhead ribozyme self-cleavage reaction, and that an ionizable 2'-proton at cleavage site is required for this conformational change. We further hypothesize that deprotonation of the cleavage-site 2'-oxygen drives this conformational change.     

Equilibrium kinetics of substrate-enzyme interactions in single crystals of cytoplasmic aspartate aminotransferase

Orthorhombic single crystals of cytoplasmic aspartate aminotransferase were examined alone or in the presence of substrates or inhibitors to quantitatively compare the interaction of ligands with the active-site chromophore between soluble and crystalline enzyme. As in enzyme solutions, equilibrium kinetic measurements can be made between substrates and single crystals of cytoplasmic aspartate aminotransferase. The absorption spectra of ligand-free enzyme forms and of enzyme-substrate or-inhibitor complexes are as distinctive as when the enzyme is in solution. The dissociation constants for glutamate with the pyridoxal form of the enzyme are identical to those in solution. The substrate analog erythro--hydroxyaspartate also binds with equal affinity to the active site in enzyme crystals as in solution; and the affinity of -ketoglutarate to bind in nonproductive complexes with the pyridoxal form of the enzyme is also unimpaired in the crystal (K _d =2 mM). In contrast to the affinity constants, the stoichiometry of the interactions does not appear to correlate to those in solution. In the presence of an amino acid plus keto acid substrates pair, the absorbance values of the enzyme-substrate complex(es) could be interpreted as for occupany of only half the available sites in the crystals. Yet an amino acid, cysteine sulfinate, and -keto acids such as , -difluorooxalacetate convert all active sites in the crystal to the pyridoxamine or pyridoxal form when added to the pyridoxal or pyridoxamine forms, respectively. This ability to completely undergo substrate-induced half-transamination and the apparently conflicting results in trapping half the sites in enzyme-substrate complexes are incorporated into a proposed reciprocating mechanism applicable only to the crystalline state of the enzyme and dictated by crystal packing forces rather than an intrinsic property of the enzyme. Active-site bound pyridoxal phosphate continues to behave as a pH indicator; nevertheless, the pK value of the single crystals is a pH unit (pK=7.15) higher than that in solution. This variation is interpreted as indication of a difference in the environment of the chromophore between the crystal and solution states. While the environmental difference does not significantly alter the affinity for substrates, it could account for the reduced rates in transformation of the enzyme-substrate complexes in half-transamination reactions in the crystalline state.     

Pyrroline 5-carboxylate dehydrogenase of the mitochondrial matrix of rat liver. Purification, physical and kinetic characteristics

The oxidation of proline to glutamate in mitochondria requires two enzymes, proline oxidase and pyrroline 5-carboxylate (P5C) dehydrogenase. In this paper we report an 800-fold purification P5C dehydrogenase from rat liver mitochondria to yield an essentially homogenous protein. The protein, whose Mr is 59,000, is an alpha 2 dimer (Mr = 115,000) in solution with an isoionic point at pH 5.7. The substrates P5C and NAD+ have apparent dissociation constants of 0.16 and 1.0 mM, respectively. Studies have been conducted to see if the conversion of glutamate and NADH to P5C and NAD+ is catalyzed by this enzyme. These studies have established that if the reverse reaction occurs the rate is 1/15,000th of the rate at which P5C is oxidized to glutamate. The concentration of the substrates needed in the assay results in a high background that interferes with accurate spectrophotometric analysis of the rate of NADH production; therefore a radiochemical (2) or a new colorimetric (3) assay was used here. A number of aldehydes were tested as substrates. It was found that the rat and human enzymes (4) have similar requirements for an aldehyde to be a substrate. Both of these proteins interacted with a polyclonal rabbit anti-rat P5C dehydrogenase serum.     

Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics

The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.     

Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 A resolution.

ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) (ATP: oxaloacetate carboxylyase (transphosphorylating), EC 4.1.1.49) is a key enzyme involved in the catabolism of glucose and amino acids in the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Due to the significant differences in the amino acid sequence and substrate specificity of the human enzyme (PEPCK (GTP-dependent), EC 4.1.1.32), the parasite enzyme has been considered a good target for the development of new anti-chagasic drugs. We have solved the crystal structure of the recombinant PEPCK of T. cruzi up to 2.0 A resolution, characterised the dimeric organisation of the enzyme by solution small angle X-ray scattering (SAXS) and compared the enzyme structure with the known crystal structure of the monomeric PEPCK from Escherichia coli. The dimeric structure possesses 2-fold symmetry, with each monomer sharing a high degree of structural similarity with the monomeric structure of the E. coli PEPCK. Each monomer folds into two complex mixed alpha/beta domains, with the active site located in a deep cleft between the domains. The two active sites in the dimer are far apart from each other, in an arrangement that seems to permit an independent access of the substrates to the two active sites. All residues of the E. coli PEPCK structure that had been found to interact with substrates and metal cofactors have been found conserved and in a substantially equivalent spatial disposition in the T. cruzi PEPCK structure. No substrate or metal ion was present in the crystal structure. A sulphate ion from the crystallisation medium has been found bound to the active site. Solution SAXS data suggest that, in solutions with lower sulphate concentration than that used for the crystallisation experiments, the actual enzyme conformation may be slightly different from its conformation in the crystal structure. This could be due to a conformational transition upon sulphate binding, similar to the ATP-induced transition observed in the E. coli PEPCK, or to crystal packing effects. The present structure of the T. cruzi PEPCK will provide a good basis for the modelling of new anti-chagasic drug leads.     

The electrode adsorption method for determination of enzyme activity: a study of substrate requirements

The electrode adsorption method for the determination of enzyme activity requires substrates that, besides having good kinetics constants for the enzyme, also show good adsorption/desorption kinetics to the electrode surface and adsorb in such a way that they change the double-layer capacitance of the electrode. A series of peptide substrates containing one to three aromatic groups has been synthesized. Our results show that the aromatic groups are of crucial importance for the capacitance change caused by the adsorbing/desorbing substrate. Thus, the tripeptide substrate, Bz-Phe(NO2)-Val-Arg-pNA, with three aromatic groups is superior to the other synthesized substrates containing only one or two aromatic groups. Our desorption experiments show that several factors determine the rate of capacitance increase observed when thrombin is added to a substrate solution in equilibrium with a substrate-covered electrode. The kinetic constants of the substrate determine how the substrate concentration in the solution decreases and, consequently, determine the spontaneous desorption measured as capacitance increase. Thrombin does not seem to split adsorbed substrate molecules but it adsorbs to the substrate-covered surface and in that way causes a capacitance decrease counteracting the change caused by desorption of substrate.     

New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine.

A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of a single amide bond converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Macroscopic kinetic constants for the interaction of these substrates with bovine trypsin, human and dog plasmin, and human thrombin are reported. Certain of these substrates exhibit extremely large specificity constants. For example, the kcat./Km for bovine trypsin with bis-(N-benzyloxycarbonylglycyl-argininamido)-Rhodamine [(Cbz-Gly-Arg-NH)2-Rhodamine] is 1 670 000 M-1 X S-1. Certain of these substrates are also highly selective. For example, the most specific substrate for human plasmin, (Cbz-Phe-Arg-NH2)-Rhodamine, is not hydrolysed by human thrombin, and the most specific substrate for human thrombin, (Cbz-Pro-Arg-NH)2-Rhodamine, is one of the least specific substrates for human plasmin. Comparison of the kinetic constants for hydrolysis of the dipeptide substrates with that of the single amino acid derivative, (Cbz-Arg-NH)2-Rhodamine, indicates that selection of the proper amino acid residue in the P2 position can effect large increases in substrate specificity. This occurs primarily as a result of an increase in kcat. as opposed to a decrease in Km and, in certain cases, is accompanied by a large increase in selectivity. Because of their high degree of sensitivity and selectivity, these Rhodamine-based dipeptide compounds should be extremely useful substrates for studying serine proteinases.     

Effect of P2' substituents on kinetic constants for hydrolysis by cysteine proteinases.

Intramolecularly quenched fluorogenic peptide substrates with the general sequence: DABCYL-Lys-Phe-Gly-Gly-Xxx-Ala-EDANS have been utilized to explore the effect of the hydrophobicity of amino acid side chains in the P2' position on the steady-state kinetic constants for papain catalyzed hydrolysis. The results demonstrate that subsite interactions between the enzyme and the peptide substrate modulate the enzyme specificity by slowing the release of the C-terminal product. This series of substrates can be used to characterize substrate specificity studies of other cysteine proteinases.     

Human carbonyl reductase 1 is an S-nitrosoglutathione reductase

Human carbonyl reductase 1 (hCBR1) is an NADPH-dependent short chain dehydrogenase/reductase with broad substrate specificity and is thought to be responsible for the in vivo reduction of quinones, prostaglandins, and other carbonyl-containing compounds including xenobiotics. In addition, hCBR1 possesses a glutathione binding site that allows for increased affinity toward GSH-conjugated molecules. It has been suggested that the GSH-binding site is near the active site; however, no structures with GSH or GSH conjugates have been reported. We have solved the x-ray crystal structures of hCBR1 and a substrate mimic in complex with GSH and the catalytically inert GSH conjugate hydroxymethylglutathione (HMGSH). The structures reveal the GSH-binding site and provide insight into the affinity determinants for GSH-conjugated substrates. We further demonstrate that the structural isostere of HMGSH, S-nitrosoglutathione, is an ideal hCBR1 substrate (Km = 30 microm, kcat = 450 min(-1)) with kinetic constants comparable with the best known hCBR1 substrates. Furthermore, we demonstrate that hCBR1 dependent GSNO reduction occurs in A549 lung adenocarcinoma cell lysates and suggest that hCBR1 may be involved in regulation of tissue levels of GSNO.