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.     

Human mitochondrial glutaredoxin reduces S-glutathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase

Glutaredoxins catalyze glutathione-dependent thiol disulfide oxidoreductions via a GSH-binding site and active cysteines. Recently a second human glutaredoxin (Grx2), which is targeted to either mitochondria or the nucleus, was cloned. Grx2 contains the active site sequence CSYC, which is different from the conserved CPYC motif present in the cytosolic Grx1. Here we have compared the activity of Grx2 and Grx1 using glutathionylated substrates and active site mutants. The kinetic studies showed that Grx2 catalyzes the reduction of glutathionylated substrates with a lower rate but higher affinity compared with Grx1, resulting in almost identical catalytic efficiencies (k(cat)/K(m)). Permutation of the active site motifs of Grx1 and Grx2 revealed that the CSYC sequence of Grx2 is a prerequisite for its high affinity toward glutathionylated proteins, which comes at the price of lower k(cat). Furthermore Grx2 was a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates. Using this novel electron donor pathway, Grx2 reduced low molecular weight disulfides such as CoA but with particular high efficiency glutathionylated substrates including GSSG. These results suggest an important role for Grx2 in protection and recovery from oxidative stress.     

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.     

Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway

Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.     

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.     

Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.

The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.     

Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity

A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product. In contrast, while nitrite considerably boosts the relative activity of human pancreatic alpha-amylase, its presence leads to changes in the electrostatic environment and active site conformations that substantially modify catalytic parameters and produce a novel acarbose rearrangement product. In particular, nitrite-substituted human pancreatic alpha-amylase demonstrates the unique ability to cleave acarbose into its acarviosine and maltose parts and carry out a previously unseen product elongation. In a completely unexpected turn of events, structural studies show that in azide-bound human pancreatic alpha-amylase, the normally resident chloride ion is retained in its binding site and an azide anion is found bound in an embedded side pocket in the substrate binding cleft. These results clearly indicate that azide enzymatic activation occurs via a mechanism distinct from that of the nitrite and nitrate anions.     

Dipeptide binding to the extended active site of the Streptomyces R61 D-alanyl-D-alanine-peptidase: the path to a specific substrate

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-D-alanine(DD)-peptidases/transpeptidases. The natural substrates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The binding of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent beta-lactam probe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the beta-lactam binding site. Hydrophobic peptides such as Gly-L-Met and Gly-L-aminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combination of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine. Steady-state kinetic measurements established this compound as the most specific peptide substrate yet discovered for a DD-peptidase by at least 3 orders of magnitude (k(cat) = 69 s(-1), K(m) = 7.9 microM, k(cat)/K(m) = 8.7 x 10(6) s(-1) M(-1)); acylation was rate-determining at saturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of other DD-peptidases.     

Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids

The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.     

Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (k(cat)), the Michaelis constant (K(M)), and the specificity constant (k(cat)/K(M)) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K(M), and k(cat)/K(M) values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K(M) values (0.93, 0.37, 0.027, and 0.00015 microM(-1) s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K(M) value for Xyl(5) (136 microM) was roughly twice as high as that for Xyl(6) (73 microM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed.     

Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.     

Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (>4MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k(cat)(app)/K(M) probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K(M) and k(cat)(app) are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k(cat)(app), possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.     

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.     

Resolution of conformational activation in the kinetic mechanism of plasminogen activation by streptokinase

Streptokinase (SK) activates plasminogen (Pg) by specific binding and nonproteolytic expression of the Pg catalytic site, initiating Pg proteolysis to form the fibrinolytic proteinase, plasmin (Pm). The SK-induced conformational activation mechanism was investigated in quantitative kinetic and equilibrium binding studies. Progress curves of Pg activation by SK monitored by chromogenic substrate hydrolysis were parabolic, with initial rates (v(1)) that indicated no transient species and subsequent rate increases (v(2)). The v(1) dependence on SK concentration for [Glu]Pg and [Lys]Pg was hyperbolic with dissociation constants corresponding to those determined in fluorescence-based binding studies for the native Pg species, identifying v(1) as rapid SK binding and conformational activation. Comparison of [Glu]Pg and [Lys]Pg activation showed an approximately 12-fold higher affinity of SK for [Lys]Pg that was lysine-binding site dependent and no such dependence for [Glu]Pg. Stopped-flow kinetics of SK binding to fluorescently labeled Pg demonstrated at least two fast steps in the conformational activation pathway. Characterization of the specificity of the conformationally activated SK.[Lys]Pg* complex for tripeptide-p-nitroanilide substrates demonstrated 5-18- and 10-130-fold reduced specificity (k(cat)/K(m)) compared with SK.Pm and Pm, respectively, with differences in K(m) and k(cat) dependent on the P1 residue. The results support a kinetic mechanism in which SK binding and reversible conformational activation occur in a rapid equilibrium, multistep process.     

Alkaline phosphatase revisited: hydrolysis of alkyl phosphates

Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound approximately 4 A apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (P(i)) in excess of its inhibition constant (K(i) approximately 1 muM). This tight binding by P(i) has affected the majority of published kinetic constants. Furthermore, binding limits k(cat)/K(m) for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k(cat)/K(m). The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK(a) of < or = 5.5 in the free enzyme. An inactivating pK(a) of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k(cat)/K(m) on the pK(a) of the leaving group follows a Br?nsted correlation with a slope of beta(lg) = -0.85 +/- 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters. The new (32)P-based assay employed herein will facilitate continued dissection of the AP reaction by providing a means to readily follow the chemical step for phosphorylation of the enzyme.     

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

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.     

A quantitative model for allosteric control of purine reduction by murine ribonucleotide reductase

The reduction of purine nucleoside diphosphates by murine ribonucleotide reductase requires catalytic (R1) and free radical-containing (R2) enzyme subunits and deoxynucleoside triphosphate allosteric effectors. A quantitative 16 species model is presented, in which all pertinent equilibrium constants are evaluated, that accounts for the effects of the purine substrates ADP and GDP, the deoxynucleoside triphosphate allosteric effectors dGTP and dTTP, and the dimeric murine R2 subunit on both the quaternary structure of murine R1 subunit and the dependence of holoenzyme (R1(2)R2(2)) activity on substrate and effector concentrations. R1, monomeric in the absence of ligands, dimerizes in the presence of substrate, effectors, or R2(2) because each of these ligands binds R1(2) with higher affinity than R1 monomer. This leads to apparent positive heterotropic cooperativity between substrate and allosteric effector binding that is not observed when binding to the dimeric protein itself is evaluated. Allosteric activation results from an increase in k(cat) for substrate reduction upon binding of the correct effector, rather than from heterotropic cooperativity between effector and substrate. Neither the allosteric site nor the active site displays nucleotide base specificity: dissociation constants for dGTP and dTTP are nearly equivalent and K(m) and k(cat) values for both ADP and GDP are similar. R2(2) binding to R1(2) shows negative heterotropic cooperativity vis-à-vis effectors but positive heterotropic cooperativity vis-à-vis substrates. Binding of allosteric effectors to the holoenzyme shows homotropic cooperativity, suggestive of a conformational change induced by activator binding. This is consistent with kinetic results indicating full dimer activation upon binding a single equivalent of effector per R1(2)R2(2).     

Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis: implication of necessity for enzyme properties

The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k(cat)/K(m), was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl-N-N'-diacetyl chitobiose or 4-methylumbelliferyl-N-N'-N'-triacetyl chitotriose or insoluble alpha-chitin substrate. By contrast, changes to substrate affinity, K(m), and turnover rate, k(cat), varied considerably for both types of chitin substrates between the full-length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full-length enzyme. The truncated mutants retained similar substrate-binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full-length chitinase molecule. Fluorescence spectroscopy indicated that both C-terminal deletion mutants retained an active folding conformation similar to the full-length enzyme. However, a CD melting unfolding study was able to distinguish between the full-length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C-terminal chitin-binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C-terminal chitin-binding region and the fibronectin (III) motif.     

Oligopeptidase B: a new type of serine peptidase with a unique substrate-dependent temperature sensitivity

Oligopeptidase B, a member of the novel prolyl oligopeptidase family of serine peptidases, is involved in cell invasion by trypanosomes. The kinetic analysis of the reactions of oligopeptidase B, which preferentially cleaves peptides at two adjacent basic residues, has revealed significant differences from the trypsin-like serine peptidases. (i) The pH dependence of k(cat)/K(m) deviates from normal bell-shaped curves due to ionization of an enzymatic group characterized by a macroscopic pK(a) of approximately 8.3. The effect of this group is abolished at high ionic strength. (ii) The second-order acylation rate constants, k(cat)/K(m), are similar with the ester and the corresponding amide substrates, suggesting that their chemical reactivity does not prevail in the rate-limiting step. The kinetic deuterium isotope effects indicate that the rate-limiting step for k(cat)/K(m) is principally governed by conformational changes. (iii) The pH-k(cat)/K(m) profile and the very low rate constant for benzoyl-citrulline ethyl ester reveal a new kinetically influential group ionizing below the pK(a) of the active site histidine and indicate that the positive charge of arginine is essential for effective catalysis. (iv) The enzyme is inhibited by high concentrations of substrate. The mechanism of inhibition markedly varies with the reaction conditions. (v) The optimum temperature for the reactions of amide substrates is unusually low, slightly below 25 degrees C, whereas with benzoyl-arginine ethyl ester a linear Eyring plot is obtained up to 39 degrees C. The positive entropies of activation point to substantial reorganization of water molecules upon substrate binding.