Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III)

A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.     

Effect of Calcium Ions on Enteropeptidase Catalysis

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.     

Substrate specificity of aminopeptidase from the mid-gut gland of the scallop (Patinopecten yessoensis)

An action for various peptides and a kinetic study for amino acid p-nitroanilides (pNAs) and 4-methylcoumaryl-7-amides (MCAs) were performed with purified aminopeptidase from the mid-gut of the scallop. The enzyme preferred dipeptides having Ala, Met, and Phe in the amino-terminal or the penultimate position from the amino-termini. The catalytic efficiencies, k(cat)/K(m) values for Ala-pNA and MCA were the highest in the tested substrates, and those for pNA and MCA substrates having Met or Phe were the next highest. The enzyme was found to be a new alanine-specific aminopeptidase.     

Steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and N-terminally truncated recombinant human methionine aminopeptidases (type 2)

The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.     

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.     

The role of Glu(192) in the allosteric control of the S(2)' and S(3)' subsites of thrombin

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir(52-65)) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S(2)' and S(3)' to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P(2)' and P(3)'. Regardless of the amino acids, Hir(52-65) decreased, and heparin increased the k(cat)/K(m) value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu(192) participates in this modulation. We have examined the role of Glu(192) by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P(2)' residue was cleaved with a k(cat)/K(m) value only 49 times higher than the one having the "least favorable" P(2)' residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P(3)' residues and its strong aversion for negatively charged P(3)' residues. Furthermore, both Hir(52-65) and heparin increased the k(cat)/K(m) value of substrate hydrolysis. We conclude that Glu(192) is critical for the P(2)' and P(3)' specificities of thrombin and for the allostery mediated through exosite 1.     

In vivo detection of aflatoxin-induced lipid free radicals in rat bile

Human kallikrein hK3 (prostate-specific antigen) is a chymotrypsin-like serine protease which is widely used in the diagnosis of prostate cancer. Assays of the enzymatic activity of hK3 in extracellular fluids have been limited by a lack of sensitive synthetic substrates. This report describes the design of a series of internally quenched fluorescent peptides containing an amino acid sequence based on preferential hK3 cleavage sites in semenogelins. Those were identified by 2-D gel electrophoresis analysis and N-terminal sequencing of semenogelin fragments generated by ex vivo proteolysis in freshly ejaculated semen. These peptides were cleaved by hK3 at the C-terminal of certain tyrosyl or glutaminyl residues with k(cat)/K(m) values of 15000-60000 M(-1) s(-1). The substrate Abz-SSIYSQTEEQ-EDDnp was cleaved at the Tyr-Ser bond with a specificity constant k(cat)/K(m) of 60000 M(-1) s(-1), making it the best substrate for hK3 described to date.     

Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity. Suggestion of a common topological specificity for functionally diverse proteases

Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with K(m) values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient "triple-helical peptidase" activity and fell into one of two categories: (k(cat)/K(m))(SSP) > (k(cat)/K(m))(THP) (thermolysin, trypsin, and MMP-13) or (k(cat)/K(m))(THP) > or = (k(cat)/K(m))(SSP) and (K(m))(SSP) > (K(m))(THP) (MMP-1 and ADAMTS-4). Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triple-helical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.     

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.     

Role of the flavin domain residues,His689 and Asn732, in the catalytic mechanism of cellobiose dehydrogenase from phanerochaete chrysosporium

Cellobiose dehydrogenase is an extracellular flavocytochrome, which catalyzes the oxidation of cellobiose and other soluble oligosaccharides to their respective lactones, while reducing various one- and two-electron acceptors. Two residues at the active site of the flavin domain, His689 and Asn732, have been proposed to play critical roles in the oxidation of the substrate. To test these proposals, each residue was substituted with either a Gln, Asn, Glu, Asp, Val, Ala, and/or a His residue by site-directed mutagenesis, using a homologous expression system previously developed in our laboratory. This enabled an examination of the functional, stereochemical, and electrostatic constraints for binding and oxidation of the substrate. The steady-state kinetic parameters for the variant proteins were compared using cellobiose and its epimer, lactose, as the substrates. The H689 variants all exhibit >1000-fold lower k(cat) values, while the K(m) values for both substrates in these variants are similar to that of the wild-type enzyme. This supports the proposed role of this His residue as a general base in catalysis. The N732 variants exhibit a range of kinetic parameters: the k(cat) values for oxidation are 5-4000-fold lower than that for the wild-type enzyme, while the K(m) values vary between similar to and 60-fold higher than that for the wild-type. The difference in binding energy between cellobiose and lactose was calculated using the relationship delta(delta G) = -RT ln[(k(cat)/K(m))(lactose)/(k(cat)/K(m))(cellobiose)]. This calculation for the wild-type enzyme suggests that lactose binds considerably more weakly than cellobiose (7.2 kJ/mol difference), which corresponds to one extra (cumulative) hydrogen bond for cellobiose over lactose. Mutations at Asn732 result in a further weakening of lactose binding over cellobiose (2-4 kJ/mol difference). The results support a role for Asn732 in the binding of the substrate.     

Human bile salt-dependent lipase efficiency on medium-chain acyl-containing substrates: control by sodium taurocholate

Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (～3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 μM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.     

Exosite interactions impact matrix metalloproteinase collagen specificities

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.     

Probing functional perfection in substructures of ribonuclease T1: double combinatorial random mutagenesis involving Asn43, Asn44, and Glu46 in the guanine binding loop

Combinatorial random mutageneses involving either Asn43 with Asn44 (set 1) or Glu46 with an adjacent insertion (set 2) were undertaken to explore the functional perfection of the guanine recognition loop of ribonuclease T(1) (RNase T(1)). Four hundred unique recombinants were screened in each set for their ability to enhance enzyme catalysis of RNA cleavage. After a thorough selection procedure, only six variants were found that were either as active or more active than wild type which included substitutions of Asn43 by Gly, His, Leu, or Thr, an unplanned Tyr45Ser substitution and Glu46Pro with an adjacent Glu47 insertion. Asn43His-RNase T(1) has the same loop sequence as that for RNases Pb(1) and Fl(2). None of the most active mutants were single substitutions at Asn44 or double substitutions at Asn43 and Asn44. A total of 13 variants were purified, and these were subjected to kinetic analysis using RNA, GpC, and ApC as substrates. Modestly enhanced activities with GpC and RNA involved both k(cat) and K(M) effects. Mutants having low activity with GpC had proportionately even lower relative activity with RNA. Asn43Gly-RNase T(1) and all five of the purified mutants in set 2 exhibited similar values of k(cat)/K(M) for ApC which were the highest observed and about 10-fold that for wild type. The specificity ratio [(k(cat)/K(M))(GpC)/(k(cat)/K(M))(ApC)] varied over 30 000-fold including a 10-fold increase [Asn43His variant; mainly due to a low (k(cat)/K(M))(ApC)] and a 3000-fold decrease (Glu46Ser/(insert)Gly47 variant; mainly due to a low (k(cat)/K(M))(GpC)) as compared with wild type. It is interesting that k(cat) (GpC) for the Tyr45Ser variant was almost 4-fold greater than for wild type and that Pro46/(insert)Glu47 RNase T(1) is 70-fold more active than the permuted variant (insert)Pro47-RNase T(1) which has a conserved Glu46. In any event, the observation that only 6 out of 800 variants surveyed had wild-type activity supports the view that functional perfection of the guanine recognition loop of RNase T(1) has been achieved.     

Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.     

A case for reverse protonation: identification of Glu160 as an acid/base catalyst in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic analysis of a site-directed mutant

The catalytic mechanism of the family 39 Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) involves a two-step double-displacement mechanism in which a covalent alpha-xylosyl-enzyme intermediate is formed with assistance from a general acid and then hydrolyzed with assistance from a general base. Incubation of recombinant XynB with the newly synthesized active site-directed inhibitor, N-bromoacetyl-beta-D-xylopyranosylamine, resulted in rapid, time-dependent inactivation of the enzyme (k(i)/K(i) = 4.3 x 10(-4) s(-1)mM(- 1)). Protection from inactivation using xylose or benzyl 1-thio-beta-xyloside suggested that the inactivation was active site-directed. Mass spectrometric analysis indicated that incubation of the enzyme with the inactivator resulted in the stoichiometric formation of a new enzyme species bearing the label. Comparative mapping of peptic digests of both the labeled and unlabeled enzyme by HPLC coupled to an electrospray ionization mass spectrometer permitted the identification of a labeled peptide. Sequencing of this peptide by tandem mass spectrometry identified Glu160 within the sequence (157)IWNEPNL(164) as the site of attachment of the N-acetyl-beta-D-xylopyranosylamine moiety. Kinetic analysis of the Glu160Ala mutant strongly suggests that this residue is involved in acid/base catalysis as follows. First, a significant difference in the dependence of k(cat)/K(m) on pH as compared to that seen for the wild-type enzyme was found, as expected for a residue that is involved in acid/base catalysis. The changes, however, were not as simple as those seen in other cases. Second, a dramatic decrease (up to 10(5)-fold) in the catalytic efficiency (k(cat)/K(m)) of the enzyme with a substrate requiring protonic assistance is observed upon such mutation. In contrast, the catalytic efficiency of the enzyme with substrates bearing a good leaving group, not requiring acid catalysis, is only moderately impaired relative to that of the wild-type enzyme (8-fold). Surprisingly, however, the glycosylation step was rate-limiting for all but the most reactive substrates. Last, the addition of azide as a competitive nucleophile resulted in the formation of a beta-xylosyl azide product and increased the k(cat) and K(m) values up to 8-fold while k(cat)/K(m) remained relatively unchanged. Such kinetic behavior is consistent with azide acting competitively with water as a nucleophile in the second step of the enzyme catalyzed reaction involving breakdown of the xylosyl-enzyme intermediate. Together, these results provide strong evidence for a role of Glu160 in acid/base catalysis but suggest that it may be partnered by a second carboxylic acid residue and that the enzyme may function through using acid catalysis involving reverse protonation of active site residues.     

Specificity of natural and artificial substrates for human Cdc25A

Cdc25A is a dual-specific protein phosphatase involved in the regulation of the kinase activity of Cdk-cyclin complexes in the eukaryotic cell cycle. To understand the mechanism of this important regulator, we have generated highly purified biochemical reagents to determine the kinetic constants for human Cdc25A with respect to a set of peptidic, artificial, and natural substrates. Cdc25A and its catalytic domain (dN25A) demonstrate very similar kinetics toward the artificial substrates p-nitrophenyl phosphate (k(cat)/K(m) = 15-25 M(-1) s(-1)) and 3-O-methylfluorescein phosphate (k(cat)/K(m) = 1.1-1.3 x 10(4) M(-1) s(-1)). Phospho-peptide substrates exhibit extremely low second-order rate constants and a flat specificity profile toward Cdc25A and dN25A (k(cat)/K(m) = 1 to 10 M(-1) s(-1)). In contrast to peptidic substrates, Cdc25A and dN25A are highly active phosphatases toward the natural substrate, T14- and Y15-bis-phosphorylated Cdk2/CycA complex (Cdk2-pTpY/CycA) with k(cat)/K(m) values of 1.0-1.1 x 10(6) M(-1) s(-1). In the context of the Cdk2-pTpY/CycA complex, phospho-threonine is preferred over phospho-tyrosine by more than 10-fold. The highly homologous catalytic domain of Cdc25c is essentially inactive toward Cdk2-pTpY/CycA. Taken together these data indicate that a significant degree of the specificity of Cdc25 toward its Cdk substrate resides within the catalytic domain itself and yet is in a region(s) that is outside the phosphate binding site of the enzyme.