Synthetic chimeras of mouse growth factor-associated glandular kallikreins. II. Growth factor binding properties.

Six chimeric constructs of the sequentially similar growth factor-associated kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and beta-NGF, analogous to the high molecular weight forms (7S NGF and HMW-EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native beta-NGF (along with native alpha-NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an alpha 2 beta gamma 2 structure (where beta-NGF is itself a dimer), with recombinant (r) gamma-NGF and with a chimera in which the amino terminal region from EGF-BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of gamma-NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from gamma-NGF retained at least some ability to form the 7S complex. rEGF-BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF-BP and the carboxyl terminal segment of gamma-NGF formed a nativelike HMW-EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and beta-NGF, respectively.     

Substrate specificities of growth factor associated kallikreins of the mouse submandibular gland

The kinetic constants for the hydrolysis of a series of tripeptide p-nitroanilide substrates by mouse epidermal growth factor binding protein (EGF-BP), the gamma-subunit of mouse nerve growth factor (gamma-NGF), bovine pancreatic trypsin (BPT), and porcine pancreatic kallikrein (PPK) have been evaluated. These substrates correspond to the carboxyl-terminal three amino acids of the mature forms of epidermal growth factor (EGF) and beta-nerve growth factor (beta-NGF), as well as various substitutions in the penultimate and antepenultimate positions, and, as such, represent potential recognition sites for precursor processing. The mouse kallikreins (EGF-BP and gamma-NGF) preferentially hydrolyze the substrates with the sequences of their specifically associated growth factors; however, the constants derived from these reactions do not account for the association constants observed with the mature growth factors, and additional significant binding interactions between EGF-BP and EGF and between gamma-NGF and beta-NGF are predicted to exist outside of the catalytic binding site, i.e., the P3 to P1 positions. A comparison of the kinetic constants of BPT, PPK, and the mouse kallikreins indicates that EGF-BP and gamma-NGF display a hybrid catalytic character. A favorable substrate P1 arginine guanidinium group interaction exists for the mouse kallikreins, similar to that of BPT, but a preference for a hydrophobic side chain in the substrate P2 position makes the mouse kallikreins, especially EGF-BP, more closely resemble PPK than BPT. These findings have significant implications with regard to molecular modeling of the mouse kallikreins.     

Prediction of the three-dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein.

We have predicted the three-dimensional structures of the serine protease subunits (gamma-NGF, alpha-NGF, and EGF-BP) of the high molecular weight complexes of nerve growth factor (7S NGF) and epidermal growth factor (HMW-EGF) from the mouse submandibular gland (from the X-ray crystal structures of two related glandular kallikreins). The conformations of three of the six loops surrounding the active site are relatively well defined in the models of gamma-NGF and EGF-BP, but three other loops are likely to have flexible conformations. Although the amino acid sequence of alpha-NGF is closely related to those of gamma-NGF and EGF-BP, it is catalytically inactive. Model-building studies on alpha-NGF suggested that mutations (in alpha-NGF) just prior to the active site serine (195) and an unusual N-terminal sequence are consistent with alpha-NGF having a zymogen-like conformation (similar to that in chymotrypsinogen). An hypothetical model of the quaternary structure of HMW-EGF has been constructed using this model of EGF-BP and the NMR structure of murine EGF. The C-terminal arm of EGF was modeled into the active site of EGF-BP based on data indicating that the C-terminal arginine of EGF occupies the S1 subsite of EGF-BP. Data suggesting one of the surface loops of EGF-BP is buried in the HMW-EGF complex and symmetry constraints were important in deriving a schematic model. A molecular docking program was used to fit EGF to EGF-BP.     

Nerve growth factor alpha subunit: effect of site-directed mutations on catalytic activity and 7S NGF complex formation

Mouse alpha- and gamma-nerve growth factor (NGF) are glandular kallikreins that form a non-covalent complex (7S NGF) with beta-NGF. gamma-NGF is an active arginine-specific esteropeptidase; the alpha-subunit is catalytically inactive and has a zymogen-like conformation. Site-directed mutagenesis of alpha-NGF to alter the N-terminus and three residues in loop 7, a region that contributes to the catalytic center, restored substantial catalytic activity against N-benzoyl arginine-p-nitroanilide as substrate in two derivatives although they were not as active as recombinant gamma-NGF. Seven of the 15 derivatives that remained more alpha-like were able to substitute for native alpha-NGF in reforming 7S complexes; the other eight derivatives that were more gamma-like showed greatly reduced ability to do so. However, the most gamma-like alpha-NGF derivative could not substitute for native gamma-NGF in 7S complex formation. These findings suggest that the alpha-NGF backbone can be corrected to a functional enzyme by the addition of a normal N-terminal structure and two catalytic site substitutions and that the 7S complex requires one kallikrein subunit in the zymogen form and one in an active conformation.     

Enkephalin hydrolysis by human serum biotinidase.

Purified human serum biotinidase exhibited amino-exo-peptidase activity. Enkephalins and dynorphin A (less than 10-mer) seemed to be the most appropriate substrates among various physiological peptides in terms of the kcat/Km values. Similar kcat/Km values were obtained for both biocytin (biotinyllysine) and these opioid-neuropeptides. Neuro-oligo-peptides ranging from 2-mer to 18-mer were hydrolyzed. The presence of amino group at the carboxyl terminal position increased the kcat/Km value by decreasing the Km value. The results of inhibition studies using various kinds of antibiotic inhibitors, metals, and chelating agents indicated that enkephalin hydrolysis was mediated by the peptide-hydrolyzing center probably containing Zn ions. This aminopeptidase activity was uniquely inhibited by a vitamin of biocytin. The reason for the high content of biotinidase activity in serum may be related to the binary function of this enzyme; i.e., biocytin hydrolyzing amidase and enkephalin hydrolyzing aminopeptidase functions.     

New substrates for enteropeptidase. I. Biologically active hepta-nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue provided that less than four negatively charged amino acid residues are in positions P2-P5 of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR decreases VYIHPF) and the Hb(2-8) (LTAEEK decreases A) and Hb(1-9) (MLTAEEK decreases AA) peptides of the cattle hemoglobin beta-chain. The Km values for all the substrates (approximately 10(-3) M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the -DDDDK- full-size linker (Km approximately 10(-4) M). The kcat values for AT and Hb(2-8) were also close and low (approximately 30 min-1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2-8) peptide by one amino acid residue from its N- or C-terminus results in a dramatic increase in the catalytic efficiency of the hydrolysis: the kcat value for Hb(1-9) is 1510 min-1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor binding protein: identification of a different protein

Partial amino acid sequence analysis of epidermal growth factor binding protein (EGF-BP), an arginine esteropeptidase that specifically associates with EGF to form a high molecular weight complex in male mouse submandibular glands, has revealed a single, distinct protein that is different from three previously reported forms of EGF-BP. This protein shows substantial sequence homology with these other putative forms of EGF-BP as well as with a large family of kallikreins expressed in the mouse submandibular gland. Purified EGF-BP contains three polypeptide chains as a result of two internal cleavages at residues 87-88 and 140-141. These modifications may represent processing events that are critical for determining the binding specificity of EGF-BP, since they occur within regions surrounding the substrate binding site.     

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.     

Intrinsic versus extrinsic coagulation. Kinetic considerations.

A study to compare the kinetics of activation of factor IX by Factor XIa/Ca2+ and by Factor VIIa/tissue factor/Ca2+ has been undertaken. When purified human proteins, detergent-extracted brain tissue factor and tritiated-activation-peptide-release assays were utilized, the kinetic constants obtained were: Km = 310 nM, kcat. = 25 min-1 for Factor XIa and Km = 210 nM, kcat. = 15 min-1 for Factor VIIa. The kinetic constants for the activation of Factor X by Factor VIIa/brain tissue factor were: Km = 205 nM, kcat. = 70 min-1. Predicted rates for the generation of Factor IXa and Factor Xa were obtained when human monocytic tumour U937 cells (source of tissue factor) and Factor VIIa were used to form the activator. In other experiments, inclusion of high-Mr kininogen did not increase the activation rates of Factor IX by Factor XIa in the presence or absence of platelets and/or denuded rabbit aorta. These kinetic data strongly indicate that both Factor XIa and Factor VIIa play physiologically significant roles in the activation of Factor IX.     

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.     

beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22

Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does the Kunitz domain from the Alzheimer's amyloid beta protein precursor inhibit a kallikrein responsible for post-translational processing of nerve growth factor precursor?

Alternative splicing of the Alzheimer's amyloid beta protein precursor (ABPP) message leads to the production of several variants of this precursor polypeptide. Two of these variants contain a domain that is highly homologous to members of the Kunitz class of protease inhibitors. In order to initiate a study of the physiological role of this domain, we have produced active ABPP Kunitz inhibitor by constructing and expressing a synthetic gene in E. coli. Nerve growth factor (NGF) deficiency has been suggested as a possible cause of the neural degeneration characteristic of Alzheimer's disease, and trypsin and gamma-NGF are the two enzymes that have been shown to be capable of processing beta-NGF precursor to active, mature beta-NGF in vitro, therefore, the specificity of purified recombinant ABPP Kunitz inhibitor was analyzed with respect to these two proteases. Binding of isolated ABPP Kunitz domain both to trypsin (Ki,app less than 10 nM and to gamma-NGF (Ki,app = 300 nM) was observed. This difference in binding to the two proteases correlates with the approximately 20-fold higher rate observed for in vitro processing of the beta-NGF precursor by trypsin compared to processing by gamma-NGF, indicating that perhaps the inhibitor mimics the interaction of the beta-NGF precursor with proteases. The kallikrein actually responsible for beta-NGF precursor processing in vivo is unknown, but these results suggest that it is capable of being significantly inhibited by exposure to the ABPP Kunitz domain.     

Comparative enzymatic studies of human renin acting on pure natural or synthetic substrates

Some of the essential structural requirements for the enzymatic reaction of pure human renin acting on pure human and rat angiotensinogen and on their synthetic tetradecapeptide substrates were investigated. The five carboxy terminal amino acids of synthetic tetradecapeptides played a significant role in substrate recognition and/or hydrolysis by human renin. Kinetic constants Km, Kcat and kcat/Km of the various human renin assays were different according to the substrate used. The presence of either an asparagine or a threonine residue in the S'4 renin subsite did not affect significantly the kinetic constant values. A tyrosine residue, rather than a histidine residue, in the S'3 renin subsite gave the best synthetic substrate studied. When tyrosine residue was present in the S'2 renin subsite an important decrease in kcat was observed. Human angiotensinogen was hydrolysed by human renin with lower Km and kcat values than those measured with human and porcine synthetic substrates, suggesting that the 3-dimensional structure of human angiotensinogen plays a key role in the hydrolysis. This finding was supported by assays performed with rat angiotensinogen, which was cleared by human renin with the same kcat value as rat tetradecapeptide, but with a 49-fold lower Km. Between human and rat angiotensinogen a kcat/Km value of only 2-fold higher has been found in the renin assay using human substrate.     

Kinetic study of carboxypeptidase P-catalyzed reaction. Pressure and temperature dependence of kinetic parameters

Detailed kinetic analyses of carboxypeptidase P-catalyzed reactions were carried out spectrophotometrically using 3-(2-furyl)acryloyl-acylated peptide substrates. The maximum kcat/Km was observed at around pH 3.5 for the synthetic peptide substrates. The kcat/Km value decreased with increasing pH, with an apparent pKa value of 4.43. However, the maximum kcat was observed at neutral pH (pH congruent to 6) and the pKa was 4.49. These apparently different pH profiles for kcat/Km and kcat of this enzyme were due to the decreasing Km value in the acid pH region. The pressure and temperature dependences of these kinetic parameters were also measured. N-Benzoylglycyl-L-phenyllactate (Bz-Gly-OPhLac) gave dependences similar to those of the peptide substrate, suggesting that there is no distinct difference in the catalytic mechanism between the peptide and the ester hydrolyses.     

Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein

A series of 14 4-nitroanilide substrates and 17 thioester substrates have been used to measure kinetic constants with porcine pancreatic kallikrein. All of the substrates have a P1 arginine residue. The 4-nitroanilide substrates consist of seven P2-glycine and seven P2-phenylalanine tripeptides. As expected from previous results, the phenylalanine series substrates were generally 100-fold 'better' than those in the glycine series. The S3 subsite was found to 'prefer' lysine or phenylalanine, whereas glutamic acid in this position was distinctly unfavourable. The thioester substrates consisted of various thioester derivatives of arginine as well as 12 dipeptides. These substrates exhibited kcat./Km values generally 1000 times higher than the P2-phenylalanine 4-nitroanilides. With the thioesters, a P2 phenylalanine or tryptophan residue yielded the best substrates, but some of the simple derivatives of arginine were nearly as good. A comparison of the kinetic constants of the thioester substrates between the porcine enzyme and human plasma kallikrein provides further evidence that these enzymes have a similar preference for bulky P2 residues, but otherwise are quite different enzymes. The thioester substrates are nearly as reactive as oxygen ester substrates such as acetylphenylalanylarginine methyl ester for the porcine enzyme [Levison & Tomalin (1982) Biochem. J. 203, 299-302; Fiedler (1983) Adv. Exp. Med. Biol. 156A, 263-274], and owing to the greater ease in assaying with the thioesters, they should find use in routine assays for the glandular kallikreins.     

Development of a high-performance liquid chromatography assay and revision of kinetic parameters for the Staphylococcus aureus sortase transpeptidase SrtA

The SrtA isoform of the Staphylococcus aureus sortase transpeptidase is responsible for the covalent attachment of virulence- and colonization-associated proteins to the bacterial peptidoglycan. Sortase utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-d-isoGlu-Lys(-Gly5)-d-Ala-d-Ala (branched Lipid II) and secreted proteins containing a highly conserved LPXTG sequence near their C termini. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly5 portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we show that reported fluorescence quenching activity assays for SrtA are subject to marked fluorescence inner filter effect quenching, resulting in prematurely hyperbolic velocity versus substrate profiles and underestimates of the true kinetic parameters kcat and Km. We therefore devised a discontinuous high-performance liquid chromatography (HPLC)-based assay to monitor the SrtA reaction employing the same substrates used in the fluorescence quenching assay: Gly5 and Abz-LPETG-Dap(Dnp)-NH2. Fluorescence or UV detection using these substrates facilitates separate analysis of both the acylation and the transpeptidation steps of the reaction. Because HPLC was performed using fast-flow analytical columns (<8min/run), high-throughput applications of this assay for analysis of SrtA substrate specificity, kinetic mechanism, and inhibition are now feasible. Kinetic analysis using the HPLC assay revealed that the kinetic parameters for SrtA with Abz-LPETG-Dap(Dnp)-NH2 are 5.5mM for Km and 0.27s-1 for kcat. The Km for Gly5 was determined to be 140microM. These values represent a 300-fold increase in Km for the LPXTG substrate and a 12,000-fold increase in kcat over literature-reported values, suggesting that SrtA is more a robust enzyme than previous analyses indicated.     

Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N

We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA. For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U. The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters. Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme. The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks). Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU. This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS)     

Horse urinary kallikrein, II. Effect of subsite interactions on its catalytic activity

The effect of secondary-subsite interactions on the catalytic efficiency of horse urinary kallikrein was studied using as substrates oligopeptides and peptidyl-4-nitroanilides with L-Arg at P1. The known secondary specificity of tissue kallikreins for hydrophobic residues at P2 was also demonstrated for horse urinary kallikrein and a higher preference of this enzyme for L-Phe over L-Leu at P2 was evident. Interaction of subsites S3 with D-Pro and D-Phe enhanced the catalytic efficiency but tripeptidyl-4-nitroanilides with acetyl-D-Pro, L-Pro and acetyl-L-Pro at P3 were no better substrates than acetyl-dipeptidyl-4-nitroanilides. The importance of the leaving group for the catalysis was proved by higher kcat/Km values for the peptides in relation to peptidyl-4-nitroanilides containing a common acyl-chain. The low kcat value for the peptide with L-Pro at P'2 stresses the importance of a hydrogen bond between P'2 amide and the carbonyl group at S'2. One L-arginine residue at the leaving group, specially at the P'2 position, decreases the value of the apparent Km. This effect resulting of side-chain interactions with S'2, is impaired by a second L-Arg at P'1.     

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.