Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases

Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.     

Properties of the His57-Asp102 dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha1-proteinase inhibitor complexed forms

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).     

Identification of a flexible loop region (297-313) of urokinase-type plasminogen activator, which helps determine its catalytic activity

Pro-urokinase has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. Lys300(c143) in an apparent "flexible loop" region (297-313) was previously shown to be an important determinant of this intrinsic catalytic activity. This was related to the loop allowing the positive charge of Lys300(c143) to transiently interact with Asp355(c194), thereby inducing an active conformation of the protease domain (Liu, J. N., Tang, W., Sun, Z., Kung, W., Pannell, R., Sarmientos, P., and Gurewich, V. (1996) Biochemistry 35, 14070-14076). To further test this hypothesis, the charge at position 300(c143) and the flexibility of the loop were altered using site-directed mutagenesis designed according to a computer model to affect the interaction between Lys300(c143) and Asp355(c194). When the charge at Lys300(c143) but not Lys313(c156) was reduced, a significant reduction in the intrinsic catalytic activity occurred. Similarly, when the flexibility (wobbliness) of the loop was enhanced reducing the size of side chain, the intrinsic catalytic activity was also reduced. By contrast, when the loop was made less flexible, the intrinsic catalytic activity was increased. These findings were consistent with the hypothesis. The effects of these mutations on two-chain activity were less and often discordant with the intrinsic catalytic activity, indicating that they can be modulated independently. This structure-function disparity can be exploited to create a more zymogenic pro-urokinase (lower intrinsic catalytic activity) with a high catalytic activity, as exemplified by two of the mutants. The changes in intrinsic catalytic activity and two-chain activity induced by the mutations were due to changes in kcat rather than Km. Some significant structure-function differences between pro-urokinase and its highly homologous counterpart, tissue plasminogen activator, were also found.     

The cytoplasmic loops between domains II and III and domains III and IV in the skeletal muscle dihydropyridine receptor bind to a contiguous site in the skeletal muscle ryanodine receptor

Excitation-contraction coupling in skeletal muscle is a result of the interaction between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR). Interactions between RyR1 and DHPR are critical for the depolarization-induced activation of Ca2+ release from the sarcoplasmic reticulum, enhancement of DHPR Ca2+ channel activity, and repolarization-induced inactivation of RyR1. The DHPR III-IV loop was fused to glutathione S-transferase (GST) or His-peptide and used as a protein affinity column for 35S-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 bound specifically to the DHPR III-IV loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. Construction of chimeras between RyR1 and RyR2 showed that amino acids Lys954-Asp1112 retained full binding activity, whereas Leu922-Phe1075 had no binding activity. The RyR1 sequence Arg1076-Asp1112, previously shown to interact with the DHPR II-III loop (Leong, P., and MacLennan, D., H. (1998) J. Biol. Chem. 273, 7791-7794), bound to DHPR III-IV loop columns, but with only half the efficiency of binding of the longer RyR1 sequence, Lys954-Asp1112. These data suggest that the site of DHPR III-IV loop interaction contains elements from both the Lys954-Phe1075 and Arg1076-Asp1112 fragments. The presence of 4 +/- 0.4 microM GST-DHPR II-III or 5 +/- 0.1 microM His-peptide-DHPR III-IV was required for half-maximal co-purification of 35S-labeled RyR1 Leu922-Asp1112 on glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of 35S-labeled RyR1 Leu922-Asp1112 binding to GST-DHPR II-III and GST-DHPR III-IV by His10-DHPR II-III and His-peptide-DHPR III-IV was observed. These studies indicate that the DHPR II-III and III-IV loops bind to contiguous and possibly overlapping sites on RyR1 between Lys 954 and Asp1112.     

His...Asp catalytic dyad of ribonuclease A: structure and function of the wild-type, D121N, and D121A enzymes

The side chains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His...Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His...Asp dyad is composed of His119 and Asp121. Previously, site-directed mutagenesis was used to show that His119 has a fundamental role, to act as an acid during catalysis of RNA cleavage [Thompson, J. E., and Raines, R. T. (1994) J. Am. Chem. Soc. 116, 5467-5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 A with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, Ndelta rather than Odelta of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3'-->5')adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2',3'-cyclic phosphate. Replacing Asp121 decreases the values of kcat/Km and kcat for cleavage by 10-fold (D121N) and 10(2)-fold (D121A). Replacing Asp121 also decreases the values of kcat/Km and kcat for hydrolysis by 10(0. 5)-fold (D121N) and 10-fold (D121A) but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His...Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.     

Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.     

Infectious disease and sociodemographic characteristics of foreign immigrants in the Central Penitentiary for Men in Barcelona

A number of thrombin mutants have been constructed to investigate the role of Trp96 and the beta-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the beta-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr(60A)Pro(60B)Pro(60C)Trp(60D) Asp(60E)Lys(60F) of the beta-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp96 --> Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen approximately 3 times more slowly than thrombin, with the two beta-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting approximately 3000- and 1300-fold more slowly, respectively. The specificity constant kcat/Km for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 microM(-1) s(-1), respectively, compared to 10 and 2.5 microM(-1) s(-1) for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant Km increased approximately 6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant kcat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the beta-insertion loop is important for thrombin activity. But the mutation of Trp96 --> Ser can compensate somewhat for the loss of binding at the beta-insertion loop. The deletion of the hydrophobic interaction between Trp96 and Pro(60B)Pro(60C) appears to decrease the stability of the beta-insertion loop, thereby causing a decrease in binding efficiency.     

Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF.VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF. VIIa reversibly inhibited with a potent (Ki=0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 A resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa.5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF.VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.     

Serial cytological assay of micronucleus induction: a new tool to predict human cancer radiosensitivity

The role of the propeptide sequence and a disulfide bridge between sites 1 and 122 in chymotrypsin has been examined by comparing enzyme activities of wild-type and mutant enzymes. The kinetic constants of mutants devoid of the Cys1-Cys122 disulfide-linked propeptide show that this linkage is not important either for activity or substrate specificity. However this linkage appears to be the major factor in keeping the zymogen stable against non-specific activation. A comparison of zymogen stabilities showed that the trypsinogen propeptide is ten times more effective than the chymotrypsinogen propeptide in preventing non-specific zymogen activation during heterologous expression and secretion from yeast. This feature can also be transferred in trans to chymotrypsinogen; i.e. the chymotrypsin trypsin propeptide chimera forms a stable zymogen.     

Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme

The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.     

Crystal structure of a complement factor D mutant expressing enhanced catalytic activity

Complement factor D is a serine protease regulated by a novel mechanism that depends on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumed to be induced by the single natural substrate, C3bB, and to result in reversible reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, and Ser215 of factor D (chymotrypsinogen numbering has been used for comparison purposes) with the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to induce substantially higher catalytic activity associated with a typical serine protease alignment of the catalytic triad residues His57, Asp102, and Ser195. These results provide a partial structural explanation for the low reactivity of "resting-state" factor D toward synthetic substrates.     

Variants of tissue-type plasminogen activator which display substantially enhanced stimulation by fibrin

Unlike most proteases, tissue-type plasminogen activator (t-PA) is secreted from cells as an active, single chain "proenzyme" whose catalytic efficiency is comparable with that of the corresponding mature, two-chain enzyme. We have previously suggested that the absence of the "zymogen triad" (Asp194-His40-Ser32; chymotrypsin numbering) contributes to this unusually high enzymatic activity of single chain t-PA. Consistent with this prediction, the single chain form of a variant of t-PA containing the zymogen triad displayed dramatically reduced activity toward synthetic substrates. Activation cleavage of this variant, however, resulted in a mature, two-chain enzyme with full catalytic activity. To further examine the functional significance of the zymogen triad, we used site-specific mutagenesis to construct a variant of t-PA, t-PA/R275E,A292S,F305H, that contained this triad but could not be converted into its two-chain form by plasmin. Characterization of this variant demonstrated that the presence of the zymogen triad specifically suppressed plasminogen activation by single chain t-PA in the absence of fibrin. In addition, these studies indicated that, like wild type t-PA, zymogen activation of this variant could be accomplished by binding to the co-factor fibrin. The combination of full activity in the presence of fibrin and reduced activity in its absence resulted in novel variants of t-PA that displayed dramatically enhanced stimulation by fibrin. While the presence of fibrin increased the catalytic efficiency of t-PA toward plasminogen by a factor of approximately 520, this stimulation factor increased to 130,000 for t-PA/R275E,A292S,F305H. Plasmin-resistant, zymogen-like variants of t-PA, therefore, may represent thrombolytic enzymes with enhanced "clot selectivity."     

Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin

Activated protein C (aPC) is an important feedback regulator of the clotting cascade. In vivo, the conversion of protein C (PC) from its zymogen to activated form is mediated primarily by thrombin bound to thrombomodulin (TM), an endothelial cell surface protein. Molecular modeling suggests residues Lys37-Lys38-Lys39 of protein C's serine protease domain reside in a surface-exposed loop (variable region 1) whose high concentration of positive charge might be involved in protein-protein interactions. In this study, we have examined the role of the conserved tribasic Lys37-39 charge center in human protein C activation. This sequence was changed to acidic by substitution with Asp37-Glu38-Asp39 (DED) and Glu37-Glu38-Glu39 (EEE), or to neutrality by substitution with Gly37-Gly38-Gly39 (GGG). These mutant PCs, expressed and purified from recombinant human 293 cells, appeared normal with regard to intracellular processing, ability to be secreted, and formation of a viable active site for tripeptidyl-p-nitroanilide substrate cleavage. For activation by free thrombin, wild-type (wt) and mutant PCs displayed equivalent activation rates, as well as identical calcium-dependent inhibition of such activation. Activation of wt-PC with a soluble TM-thrombin complex yielded a 2,000-fold faster rate compared with that by free thrombin at the same (physiological) calcium level. In contrast, the acidic mutants DED and EEE exhibited virtually no TM-mediated increase in activation rate, while the neutral mutant GGG was somewhat intermediate with a 30-fold stimulation of activation rate. These reductions in activation rate were independent of the presence of chondroitin sulfate on TM. Our observations represent the first identification of residues whose mutation essentially uncouples activation by the TM-thrombin complex without affecting activation by free thrombin. Further, our results suggest that VR1 residues within the zymogen form of a serine protease can be important for recognition by physiological activators.     

Mutational analysis of Arg197 of rat surfactant protein A. His197 creates specific lipid uptake defects

In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. We substituted Ala, Lys, His, Asp, and Asn mutations for Arg to evaluate the role of a basic amino acid at position 197 in SP-A action. Unexpectedly, Ala197 retained complete activity in the SP-A functions of carbohydrate binding, type II cell binding, inhibition of surfactant secretion, lipid binding, lipid aggregation, and lipid uptake by type II cells. The results unambiguously demonstrate that Arg197 is not mechanistically essential for SP-A function. The Lys197 mutation displayed all functions of the wild type protein but exhibited a 2-fold increase in lipid uptake activity. The His197 mutation displayed all SP-A functions studied except for lipid uptake. The results obtained with the His197 mutation clearly demonstrate that lipid aggregation alone by SP-A is insufficient to promote lipid uptake by type II cells. These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes.     

Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae. Identification of additional amino acid residues involved in its catalytic activity

Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p. Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme. Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S. cerevisiae Chs1p and Chs2p. Alanine substitutions for the conserved amino acids in con2 identified five amino acids, Asn797, His799, Asp800, Trp803, and Thr805, the mutation of which severely diminished enzymatic activity and the enzyme's ability to rescue the yeast chs2 delta chs3 delta null mutant strain. Although the activities of some of the mutant enzymes were too low to measure enzyme kinetics, most of the alanine mutations in con2 affected the kcat values rather than the K(m) values. Whereas a conservative mutation of Asn797 restored the activity, those of His799, Asp800, Trp803, and Thr805 did not. A fine alignment of the amino acid sequences of con2 and Chs3p revealed that Asp800, Trp803 and Thr805 are completely conserved near the C-terminal ends of Chs3p and its homologs in other fungi. On the basis of these findings, we propose that Asp800, Trp803, and Thr805 in con2 are additional residues involved in catalysis, and hypothesise that Asp800 together with the previously identified Asp441 and Asp562 serve as polar residues necessary for the acid-based catalytic reaction of chitin synthase.     

Mapping the charged residues in the second immunoglobulin-like domain of the vascular endothelial growth factor/placenta growth factor receptor Flt-1 required for binding and structural stability

Flt-1 is one of two receptor tyrosine kinases through which the angiogenic factor vascular endothelial growth factor (VEGF) functions. Placenta growth factor (PlGF) is an additional ligand for Flt-1. The second immunoglobulin-like domain in the extracellular domain of Flt-1 has previously been identified as the region containing the critical ligand-binding determinants. We analyzed the contribution of charged residues within the first three domains of Flt-1 to ligand binding by alanine-scanning mutagenesis. Domain 2 residues Arg159, Glu208 and His223-Arg224 (together) affect both VEGF and PlGF binding, while Glu137, Lys171, His223, and Arg224 affect PlGF but not VEGF. Several charged residues, especially Asp187, are important in maintaining the structural integrity of domain 2. In addition, some residues in domain 3 contribute to binding (Asp231) or provide for additional discrimination between ligands (Arg280-Asp283).     

Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.     

Building a relationship: communications and relationships between staff and stroke patients on a rehabilitation ward

Pancreatic enzyme storage and secretion were studied in rats treated twice daily with s.c. injections (5 micrograms/kg) of CCK-8 for 3, 7, and 15 days. Isolated zymogen granules were analyzed by flow cytometry to determine their FSC (forward scatter), SSC (side scatter), and amylase and trypsinogen contents. DNA content, pancreatic weight, and both basal and stimulated pancreatic secretion under i.v. CCK infusion (1.25 micrograms/kg/h) were also studies. Two subsets of zymogen granules were identified by flow cytometry in both control and CCK-treated rats on the basis of FSC and SSC parameters: Z1 (smaller and less complex) and Z2. Both subsets displayed a high degree of heterogeneity with respect to their enzyme content per zymogen granule. During the first 7 days of CCK treatment, hyperplasia and hypertrophy developed in the rats together with changes in the zymogen granules, reflected by a significantly decreased FSC, and increased SSC, and an increase in the mean trypsinogen/amylase ratio per granule. A rise in pancreatic enzyme secretion, especially of trypsin, was observed. After 15 days of CCK administration, a simultaneous decrease in amylase content and increase in trypsinogen content per zymogen granule was observed. A desensitization of the pancreas to CCK happened after 15 days of CCK administration, reflected by a reduction of all the pancreatic functions that had been increased at shorter CCK administration periods. Nevertheless, trypsinogen appeared resistant to desensitization because its secretion significantly increased in response to an i.v. infusion of CCK. CCK treatment displayed a differential packaging of the enzymes in individual zymogen granules; the trypsinogen/amylase ration was significantly higher in Z2 zymogen granules than in Z1 subset throughout the treatment.     

Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system

A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46-51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the kcat/Km value) 100% higher than that of the wild type at 10 degrees C when N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased Km value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60 degrees C and circular dichroism spectra at 10 degrees C.     

Substrate-binding and catalytic roles of Lys194 in the C-terminus in human adenylate kinase by site-directed random mutagenesis

Site-directed random mutagenesis of Lys194 residue in the C-terminus of human adenylate kinase (AK) was performed, and six mutants were analyzed by steady-state kinetics. K194-mutants variously affected the apparent Michaelis constants (K(m) values) for ATP and AMP, although the kcat values strikingly decreased. The Lys194 residue appears to interact not only with MgATP2- but also with the AMP2- substrates by salt bridge formation with a nucleotide and to play a functional role in stabilizing the phosphate-transfer during catalysis. Lys194 could be essential for substrate-holdings and in catalysis and not replaceable to the other amino acids.