Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products.

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.     

Intersubunit electrostatic complementarity in the RecA nucleoprotein filament regulates nucleotide substrate specificity and conformational activation

The Escherichia coli RecA protein is the prototypical member of a family of molecular motors that transduces ATP binding and hydrolysis for mechanical function. While many general mechanistic features of RecA action are known, specific structural and functional insights into the molecular basis of RecA activation remain elusive. Toward a more complete understanding of the interdependence between ATP and DNA binding by RecA, we report the characterization of a mutant RecA protein wherein the aspartate residue at position 100 within the ATP binding site is replaced by arginine. Physiologically, D100R RecA was characterized by an inducible, albeit reduced, activation of the SOS response and a diminished ability to promote cellular survival after UV irradiation. Biochemically, the D100R substitution caused a surprisingly modest perturbation of RecA-ATP interactions and an unexpected and significant decrease in the affinity of RecA for ssDNA. Moreover, in vitro assays of RecA activities requiring the coordinated processing of ATP and DNA revealed (1) a 2-5-fold decrease in steady-state turnover of ATP; (2) no formation of mixed nucleoprotein filaments when wild-type and D100R RecA compete for limiting ssDNA; and (3) no formation of strand exchange reaction products. Taken together, these results suggest that the D100R mutational effects on isolated RecA activities combine synergistically to perturb its higher-order functions. We conclude that the replacement of Asp100 resulted in a change in the electrostatic complementarity between RecA monomers during active filament assembly that prevents the protein from fully accessing the active multimeric state.     

Amidation of growth hormone releasing factor (1-29) by serine carboxypeptidase catalysed transpeptidation

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1-29)-NH2, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1-29)-NH2 is -Met-Ser-Arg-NH2 the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH2----Bz-Met-Ser-Arg-NH2 + H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72% could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1-28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusive and electrostatic effects with insolubilized enzymes subject to substrate inhibition

The kinetics of an insolubilized substrate inhibited enzyme have previously been shown to lead to the possibility of multiple steady states. This paper examines the precise range of conditions under which multiple steady states will occur taking into account all combinations of the diffusive and electrostatic effects, and sample calculations are given. These calculations suggest that multiple steady states will be, in general, unusual and, within the living cell, unlikely.     

Purification of gamma-glutamyltransferase by phenyl boronate affinity chromatography. Studies on the acceptor specificity of transpeptidation by rat kidney gamma-glutamyltransferase

gamma-Glutamyltransferase has been purified from rat kidney by a novel procedure using phenyl boronate affinity chromatography. The highly purified enzyme has been studied with respect to acceptor specificity for a number of amino acids, amino acid analogues, dipeptides and tripeptides. The acceptor activity is specific for L-amino acids. The amino acids and the majority of the essential amino acids are poor acceptors while the sulphur-containing amino acids are the best acceptors. The acceptor activity is modulated by the substitution of the amino acid side chain. Substitution of the side chain at the delta, gamma or beta positions results in a proportionally decreasing ability to act as acceptor. The carbonyl moiety of the gamma-carboxy group of the acceptor appears to be essential for acceptor activity, absence of an alpha-carboxy carbonyl group increases the Kappm of the acceptor approximately 100-fold.     

Violaxanthin de-epoxidase. Lipid composition and substrate specificity.

Violaxanthin de-epoxidase isolated from lettuce chloroplasts (Lactuca sativa var. Romaine) contained a single lipid component, monogalactosyldiglyceride (MG) at about 8 g per 100 g protein. The effects of MG on activation of solvent-extracted enzyme and on K_m suggest that MG has two roles, namely, as a functional component of the binding site and as a substrate-solubilizing agent whose structure satisfies binding site requirements. Substrate specificity examined with various naturally occurring and semisynthetic epoxy carotenoids with known chirality showed violaxanthin de-epoxidase to be stereospecific for 3-hydroxy, 5,6-epoxy carotenoids which are in a 3S, 5R, 6S configuration. Although monoepoxides with the above configuration were active, their rates varied, apparently due to the influence of structural differences in the nonepoxide end groups. Hence while all-trans neoxanthin showed low rates, the de-epoxidation rate of antheraxanthin was 5-fold higher than violaxanthin. Neoxanthin and violeoxanthin, both naturally occurring pigments with 9-cis configurations in the acyclic polyene chain, were inactive. These effects support the view that violaxanthin de-epoxidase is a mono de-epoxidase and that the stereospecific active center is situated in a narrow well-like cavity which favors an all-trans configuration of the polyene chain. The 3-hydroxy, 5,6-epoxy group of the naturally occurring pigments, diadinoxanthin, antheraxanthin, and β-cryptoxanthin epoxide are assumed to be the 3S, 5R, 6S configuration based on their reactivity with violaxanthin de-epoxidase.     

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase.

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 +/- 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 - 43 000, as determined by gel electrophoresis, gel filtration in guanidine-hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine-hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5'-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent, The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported. The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.     

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.     

Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate concentration and low ionic strength.

Hyaluronidase and high levels of hyaluronan are found together in tumours. It is highly likely that hyaluronidase activity controls the balance between high molecular mass hyaluronan and oligosaccharides, and thus plays an important role in cancer development. The hyaluronan hydrolysis catalysed by bovine testicular hyaluronidase was studied as a model. The kinetics was investigated at pH 5 and 37 degrees C using the colorimetric N-acetyl-d-glucosamine reducing end assay method. While the substrate dependence obtained in the presence of 0.15 mol L(-1) ionic strength exhibited a Michaelis-Menten behaviour, an atypical behaviour was observed under low ionic strength: for increasing hyaluronan concentrations, the initial reaction rate increased, reached a maximum and then decreased to a very low level, close to zero at high substrate concentrations. One of the various hypotheses examined to explain this atypical behaviour is the formation of non-specific complexes between hyaluronan and hyaluronidase based on electrostatic interactions. This hypothesis is the only one that can explain all the experimental results including the variation of the reaction medium turbidity as a function of time and the influence on the initial reaction rate of the hyaluronan concentration over hyaluronidase concentration. However, phenomena such as the high viscosity of highly concentrated hyaluronan solutions or the steric exclusion of hyaluronidase from hyaluronan solutions may contribute to the atypical behaviour. Finally, the biological implications of the non-linear and non-monotonous shape of the hyaluronan-hyaluronidase substrate dependence in the regulation of the hyaluronan chain molecular mass are discussed, in particular in the case of cancer development.     

Pressure effects on proteolysis catalysed by calpain

The effects of pressure on mu and m-calpain stability and specific activity have been examined. Activity and stability of these neutral calcium-dependent heterodimeric proteinases were studied using an in-house built bioreactor allowing on-line spectrophotometric monitoring with retention of pressure. Both isozymes were founded to be rather baro-sensitive with t1/2 at 1500 bar of 6 min and 11 min for mu and m-calpain respectively. Activity measurements under pressure showed a biphasic behavior for both proteinases with a slight activation for pressure up to 500 bar and 750 bar for m and mu-calpain respectively. Activation volume changes indicated that the proteolytic reaction was alternatively favored and disfavored by pressure due to catalytic step activation associated with enzyme-substrate binding step being continuously inhibited by pressure. Furthermore, autoproteolysis of calpain, a calcium dependent phenomenon was inhibited by application of pressure indicating that pressure inhibition of proteolytic activity could also be due to Ca2(+)-binding decrease under pressure. Implication of these results with catalytic mechanism of these heterodimeric proteinases is also discussed.     

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Study of substrate specificity of human aromatase by site directed mutagenesis.

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation. The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15-20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate-enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.