Structural features of glutamine substrates for transglutaminases. Role of extended interactions in the specificity of human plasma factor XIIIa and of the guinea pig liver enzyme

Amino acid residues at several locations in close primary vicinity to a substrate glutamine residue have been recognized as important determinants for the specificities of human plasma factor XIIIa and guinea pig liver transglutaminase (Gorman, J. J., and Folk, J. E. (1981) J. Biol. Chem. 256, 2712-2715). The present studies measure the influence on transglutaminase specificity of some changes in amino acid side chains in a small synthetic glutamine peptide amide, Leu-Gly-Leu-Gly-Gln-Gly-Lys-Val-Leu-GlyNH2, which was designed to contain most of the known elements needed for enzyme recognition. The results are in agreement with previous findings and show that full catalytic activity of each enzyme may be retained upon replacement of the lysine residue by certain other amino acid residues. Evidence is provided that serine in place of glycine at one or more positions causes a significant increase in specificity with factor XIIIa, but not with liver enzyme. The effective substrate property for factor XIIIa seen with the model peptide amide is lost upon reversal of the sequence Val-Leu. This is not the case with the liver enzyme even though replacement of either of these amino acids by alanine causes a pronounced loss in activity with this enzyme. These differences and the effects of various other substitutions in the model peptide amide on the enzymes' specificities points up the relatively stringent structural requirements of factor XIIIa and the rather broad requirements for liver transglutaminase.     

Structural features of glutamine substrates for human plasma factor XIIIa (activated blood coagulation factor XIII)

The action of human plasma factor XIIIa (thrombin-activated blood coagulation factor XIII) and guinea pig liver transglutaminase on purified caseins, fibrin, the derivatized gamma chain of fibrin, and a number of synthetic glutamine peptides, and peptide derivatives is reported. There are wide variations in the properties of the individual proteins and peptides as substrates for amine incorporation by the two transglutaminases. beta-Casein and several of its derivatives are excellent substrates for factor XIIIa. However, beta-casein is a relatively poor substrate for the liver enzyme. The primary site of amine incorporation by factor XIIIa in beta-casein was identified as glutamine 167. This was accomplished by labeling with fluorescent amine followed by proteolytic digestion and identification of labeled peptides. An 11-residue peptide and a 15-residue peptide, each containing 1 glutamine residue and each modeled after the primary site of amine incorporation in beta-casein, were prepared. A 13-residue peptide modeled after the primary crosslinking site in fibrin gamma chain was also prepared. Each of these polypeptides proved to be an efficient substrate for factor XIIIa and displayed significantly better substrate properties than a number of small glutamine peptide derivatives that are good substrates for liver transglutaminase.     

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.     

A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.

A peptide derived from p34cdc2, cdc2(6-20)NH2 with the amino acid sequence of KVEKIGEGTYGVVYK-amide, was found to be a specific and efficient substrate for a pp60c-src-related protein tyrosine kinase from bovine spleen (STK). Glu-12 and Thr-14 were identified to be substrate specificity determinants in this peptide (Cheng, H.-C., Litwin, C. M. E., Hwang, D. M., and Wang, J. H. (1991) J. Biol. Chem. 266, 17919-17925). In this study, we demonstrated the presence of cdc2(6-20)NH2 peptide tyrosine kinase activity in the membrane fractions of bovine brain, spleen, thymus, lung, liver, and kidney. Hydroxylapatite column chromatography of thymus membrane extract revealed four protein tyrosine kinases, TK-I, TK-II, TK-III, and TK-IV, with different relative activities toward cdc2(6-20)NH2 and a general tyrosine kinase substrate, poly(Glu/Tyr). Only TK-I and TK-II showed significant activity toward cdc2(6-20)NH2, they were suggested as belonging to the src-family by virtue of their cross-reactivity with an antibody against a synthetic peptide corresponding to a conserved sequence of src-family kinases. Further immunological characterization using antibodies specific to individual src-related protein tyrosine kinases suggested that TK-I, TK-II, and STK are bovine homologs of p56lck, p55fyn, and p56lyn, respectively. Substrate specificity and kinetic characterization of src-family tyrosine kinases including human platelet pp60c-src, bovine p56lyn, p56lck, and p55fyn, as well as several non-src-related tyrosine kinases including epidermal growth factor receptor, p43v-abl, TK-III, and TK-IV showed that all the src-family tyrosine kinases but none of the other kinases displayed efficient cdc2(6-20)NH2 phosphorylation. In all cases, the high efficiency of cdc2(6-20)NH2 peptide phosphorylation could be markedly attenuated when Glu-12 and Thr-14 of the peptide were substituted, respectively, by valine and serine.     

Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2

Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several P1-P1' pseudodipeptidyl-modified substrate analogs. Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-1 enzyme (Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (1990) J. Biol. Chem. 265, 408-413). However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond. The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides. Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity. For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template. Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and P1', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions. In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease. Relative to P1-P1' statine or Phe psi [CH2N]Pro-modified pseudopeptidyl inhibitors, compounds having Xaa psi[CH(OH)CH2]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-1 protease than to the HIV-2 enzyme. Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of rat pancreatic elastase

Proelastase has been purified to homogeneity from rat pancreatic tissue by a combination of CM-Sephadex and immobilized protease inhibitor affinity resins. Trypsin activation yields an elastolytic enzyme that possesses a specificity toward small hydrophobic residues in synthetic amide substrates, similar to those of porcine elastase 1 and canine elastase. However, the rat enzyme also rapidly hydrolyzes a substrate containing tyrosine in the P1 position. N-Terminal sequence analysis reveals that rat proelastase has an identical activation peptide with that of porcine proelastase 1 and has two conservative amino acid sequence differences from the activation peptide of canine proelastase. The sequence data established that rat proelastase corresponds to the elastase 1 mRNA clone isolated by MacDonald et al. [MacDonald, R. J., Swift, G. H., Quinto, C., Swain, W., Pictet, R. L., Nikovits, W., & Rutter, W. J. (1982) Biochemistry 21, 1453]. The sequence and substrate data obtained for rat and canine elastases suggest that there is a family of pancreatic elastases with properties similar to those of the classically described porcine elastase 1.     

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.     

Rat liver polysome N alpha-acetyltransferase: substrate specificity.

The substrate specificity of polysome rat liver N alpha-acetyltransferase (NAT) has been examined by utilizing a series of synthetic and natural substrates that has been systematically altered with respect to N-terminal sequence and length. Families of peptides of the structure S-Y-S-G-G-L-L-L were generated by successively replacing the N-terminal serine, the penultimate tyrosine, and the antepenultimate serine with all 19 commonly occurring amino acids, which were then assessed for their reactivity with the rat liver enzyme. Only peptides with N-terminal serine, alanine, methionine, leucine, and phenylalanine were modified. Glycine, lysine, arginine, valine, isoleucine, and tryptophan in the second position are (with N-terminal serine) strongly inhibitory, and proline completely blocks modification. Third-position substitutions have less of an effect on NAT activity with glycine, aspartic acid, glutamic acid, and tryptophan being most inhibiting (with N-terminal Ser-Tyr). These observations are generally in agreement with in situ modifications although there are some significant differences particularly with respect to the amino-terminal residues. Optimal chain length was determined to be 10-11 residues with either synthetic peptides of the structure S-Y-S-(G)n-L-L-L or adrenocorticotropin (ACTH) sequences ranging from 8 to 39 residues. The ACTH peptides were generally found to be severalfold better substrates than the corresponding synthetic ones. Activity was not affected by increased chain length beyond approximately 17 residues. These data support the view that polysome-catalyzed N alpha-acetylation occurs as a cotranslational event on nascent chains of about 20-40 amino acids in length.     

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

Abstract Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1'. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.     

Identification of factor XIIIA-reactive glutamine acceptor and lysine donor sites within fibronectin-binding protein (FnbA) from Staphylococcus aureus

Staphylococcal fibronectin-binding protein (FnbA) is a surface-associated receptor responsible for the reversible binding of bacteria to human fibronectin and fibrin(ogen). Recently we have shown that FnbA serves as a substrate for coagulation factor XIIIa and undergoes covalent cross-linking to its ligands, resulting in the formation of heteropolymers (Matsuka, Y. V., Anderson, E. T., Milner-Fish, T., Ooi, P., and Baker, S. (2003) Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: Evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin, Biochemistry 42, 14643-14652). Factor XIIIa also catalyzes the incorporation in FnbA of fluorescent probes dansylcadaverine and glutamine-containing synthetic peptide patterned on the NH(2)-terminal segment of fibronectin. In this study, the above probes were utilized for site-specific labeling and identification of reactive Gln and Lys residues targeted by factor XIIIa in rFnbA. Probe-decorated rFnbA samples were subjected to trypsin or Glu-C digestion, followed by separation of labeled peptides using reversed phase HPLC. Sequencing and mass spectral analyses of isolated probe-modified peptides have been employed for the identification of factor XIIIa-reactive Gln and Lys residues. Analysis of dansylcadaverine-labeled peptides resulted in the identification of one major, Gln103, and three minor, Gln105, Gln783, and Gln830, amine acceptor sites. The labeling procedure with dansyl-PGGQQIV probe revealed that Lys157, Lys503, Lys620, and Lys762 serve as amine donor sites. The identified reactive glutamine acceptor and lysine donor sites of FnbA may participate in transglutaminase-mediated cross-linking reactions resulting in the covalent attachment of pathogenic Staphylococcus aureus to human host proteins.     

Phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase of synthetic peptide analogs of a site phosphorylated in histone H2B

Analogs of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B were used to assess the substrate specificity of cGMP-dependent protein kinase. cGMP-dependent kinase phosphorylated the oligopeptide Arg-Lys-Arg-Ser32-Arg-Lys-Glu with favorable kinetic parameters as compared to those for cAMP-dependent kinase (Glass, D. B., and Krebs, E. G. (1979) J. Biol. Chem. 254, 9728-9738). The contribution of each amino acid to the ability of the peptide to be phosphorylated by cGMP-dependent or cAMP-dependent kinase was studied by replacement of individual residues and evaluation of the kinetic constants of the substituted peptides. Peptides containing acetylated lysine residues or nitroarginine residues were poor substrates for both kinases. Substitution of either arginine 29 or lysine 30 with alanine increased the Km values and decreased the Vmax values for both kinases. Substitution of lysine 34 with alanine increased the Vmax values for both kinases but did not affect the Km values for either enzyme. Substitution of the phosphorylatable serine with a threonine residue greatly depressed the Vmax for both kinases. Peptides in which arginine 31 or arginine 33 were replaced by an alanine residue revealed several apparent differences in the specificity requirements between cGMP-dependent and cAMP-dependent kinases.     

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.     

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.     

Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein

The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.     

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.     

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.     

Vitamin K-dependent carboxylase: requirements for carboxylation of soluble peptide and substrate specificity.

Rat liver microsomes contain a triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of precursor proteins to γ-carboxyglutamyl residues. This activity has been studied utilizing synthetic peptides as substrates for the enzyme. When compared to the carboxylation of the endogenous microsomal precursors, the peptide carboxylase activity is more sensitive to the action of various inhibitors, and requires a higher concentration of vitamin K for maximal activity. The apparent K_m for the peptide Phe-Leu-Glu-Glu-Leu was found to be 4 mM. Substrate specificity depends on residues adjacent to the carboxylated Glu residues and macromolecular recognition sites.     

Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.     

Studies on Mold Proteases

The substrate specificity of the crystalline acid protease obtained from Rhizopus chinensis was determined using B-chain of oxidized beef insulin and numerous synthetic peptides, comparing with that of several acid proteases from various sources. The peptide bonds susceptible to the action of Rhiz. acid protease were found to be mainly those involving the amino group of bulky amino acids. The enzyme split the B-chain of oxidized insulin at twelve sites of the peptide linkages and a certain similarity in the specificity was observed among the three acid proteases, Rhiz. protease, rennin and pepsin, all of which were known to show potent milk clotting activities.