Barley alpha-amylase Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and is critical for activity and substrate specificity.

Met53 in barley alpha-amylase 1 (AMY1) is situated at the high-affinity subsite -2. While Met53 is unique to plant alpha-amylases, the adjacent Tyr52 stacks onto substrate at subsite -1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5-0.01 and 1.7-0.02% of wild-type, respectively. Km increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl alpha-d-maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite -2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild-type released 4-nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5. Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation - promoted probably by slow substrate hydrolysis - to produce up to maltoundecaose from PNPG6.     

Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme*

Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate‐type (i‐type) lyzozyme. These mutations restored the bell‐shaped pH‐dependency of the enzyme activity from the sigmoidal pH‐dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N‐acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.     

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane

The recent availability of the SHV-1 beta-lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta-lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser, Asp179Asn, Arg164His, and Arg164Ser; an inhibitor resistant group: Arg244Ser, Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyimino-cephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants: Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta-lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K(m) value for cephaloridine and oxyimino-cephalosporins compared to SHV-1 and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta-lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta-lactam resistance in this family of class A beta-lactamases.     

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin from Thiobacillus ferrooxidans: insights into the structural relationship with the cupredoxins and the multi copper proteins

The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.     

Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.     

Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase

Sixteen residues in stalk segment S5 of the Ca(2+)-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca(2+) binding transition, determined at 0 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala. In mutant Arg(762) --> Ile, the rate of the Ca(2+) binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 20-fold in the absence of K(+)) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(750) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ala, Tyr(754) --> Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca(2+) dissociation was 11-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca(2+) binding pocket and the catalytic domain and that Arg(751) is important for both structural and functional integrity of the enzyme.     

The second coordination sphere of FIH controls hydroxylation

The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) within the α-subunit of the hypoxia inducible factor (HIF). FIH is an α-ketoglutatrate (αKG)-dependent, non-heme Fe(II) dioxygenase, in which Fe(II) is coordinated by a (His(2)Asp) facial triad, αKG, and H(2)O. Hydrogen bonding among the facial triad, the HIF-Asn(803) side chain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center. Point mutants of FIH were prepared to test the functional role of the αKG-centered (Asn(205) and Asn(294)) or HIF-Asn(803)-centered (Arg(238) and Gln(239)) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O(2), oxidative decarboxylation, and substrate positioning. Steady-sate kinetics were used to test for overall catalytic effects; autohydroxylation rates were used to test for priming and positioning, and electronic spectroscopy was used to assess the primary coordination sphere and the electrophilicity of αKG. Asn(205) → Ala and Asn(294) → Ala mutants exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue-shifted metal to ligand charge transfer transitions for (Fe+αKG)FIH indicated that these point mutations destabilized the π* orbitals of αKG, further supporting a slowed rate of oxidative decarboxylation. The Arg(238) → Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; the Arg(238) → Met mutant was capable of O(2) activation for the autohydroxylation reaction. The Gln(239) → Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to the Gln(239) → Asn mutant stimulated autohydroxylation, it is more likely that this point mutant simply mispositions the HIF-Asn(803) side chain. This work combines kinetics and spectroscopy to show that these second-sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O(2), and positioning HIF-Asn(803).     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Nuclear localization of mitogen-activated protein kinase kinase 1 (MKK1) is promoted by serum stimulation and G2-M progression. Requirement for phosphorylation at the activation lip and signaling downstream of MKK

Stimulation of mammalian cells results in subcellular relocalization of Ras pathway enzymes, in which extracellular signal-regulated protein kinases rapidly translocate to nuclei. In this study, we define conditions for nuclear localization of mitogen-activated protein kinase kinase 1 (MKK1) by examining effects of perturbing the nuclear export signal (NES), the regulatory phosphorylation sites Ser218 and Ser222, and a regulatory domain at the N terminus. After disrupting the NES (Delta32-37), nuclear uptake of MKK was enhanced when quiescent cells were activated with serum-phorbol 12-myristate 13-acetate or BXB-Raf-1 cotransfection. Uptake was enhanced by mutation of Ser218 and Ser222 to Glu and Asp, respectively, and blocked by mutation of these residues to Ala, although mutation of Lys97 to Met, which renders MKK catalytically inactive, did not interfere with uptake. Therefore, nuclear uptake of MKK requires incorporation of phosphate or negatively charged residues at the activation lip but not enzyme activity. On the other hand, uptake of an active MKK mutant with disrupted NES (Delta32-51) was elevated in quiescent as well as stimulated cells, and pretreatment of cells with the MKK inhibitor 1,4-diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio]butadiene blocked nuclear uptake. Thus, signaling downstream of MKK is also necessary for translocation. Finally, wild type MKK containing an intact NES translocates to nuclei during mitosis before envelope breakdown. Comparison of mutants with Ser to Glu and Asp or Ala substitutions indicates that Ser phosphorylation is also required for mitotic nuclear uptake of MKK.     

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.     

Catalytic mechanism of phosphorylase kinase probed by mutational studies.

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.     

Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase

The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein. The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein. In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala. Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time. High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus. The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants. These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase.     

Recombinant chicken egg white cystatin variants of the QLVSG region.

Using recombinant DNA methods, seven cystatin variants were produced by cassette mutagenesis of a chicken egg white cystatin variant which already contains the mutations Ala3, Glu2, Phe1, Ser1-->Met, Met29-->and Met 89-->Leu. When characterized by structural and functional studies, they were all found to harbour mutations in the first hairpin loop, the so-called 'QXVXG' region, which is highly conserved within the cystatin superfamily and thought to be important for its inhibitory activity towards cysteine proteinases. They were purified to more than 90% homogeneity and analysed by SDS/PAGE, HPLC, tryptic peptide mapping, N-terminal amino acid sequencing and ELISA. Structural model building of the variants and their complexes with papain was performed using computer graphics based on the crystallographic coordinates of chicken egg white cystatin and the papain-stefin complex. Only minor conformational changes were required for modelling the mutants or complexes. Equilibrium dissociation constants and rate constants of complex formation of the variants with papain, actinidin as well as cathepsin B and L were determined by kinetic measurements using fluorogenic substrates. The single exchanges Gln53-->Glu, Gln53-->Asn, Val44-->Asp, Gly57-->Ala and the double exchanges Arg52-->Leu, Gln53-->Glu, Gln53-->Asn, Ser56-->Ala, Leu54-->Met, Gly57-->Ala reduced the inhibition of papain, actinidin and cathespin B significantly by 10-1000-fold. With the exception of the Val55-->Asp variant, the differences in the Ki values are mainly due to larger k off values, whereas the kon values seem to be more or less unaffected by the selected mutations. The effect on the inhibition of papain is generally smaller than the effects on actinidin and cathepsin B inhibition. Cathepsin L inhibition is strikingly insensitive to all mutations. These distinct effects of the inhibitor variants indicate differences in proteinase-inhibitor-protein interactions between closely related cysteine proteinases. In addition, the results verify the prediction, made earlier from sequence alignment studies and from a docking model of the chicken cystatin-papain complex, that the first hairpin loop of cystatins is essential for effective inhibition.     

Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl.

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Mutants at Ser277 of Xenopus cdc2 protein kinase induce oocyte maturation in the absence of the positive regulatory phosphorylation site Thr161.

The cdc2 protein kinase is an important regulatory protein for both meiosis and mitosis. Previously, we demonstrated that simultaneous mutation of Thr14-->Ala14 and Tyr15-->Phe15 in the Xenopus cdc2 protein results in an activated cdc2 mutant that induces maturation in resting oocytes. In addition, we confirmed the importance of the positive regulatory phosphorylation site, Thr161, by demonstrating that cdc2 mutants containing additional mutations of Thr161-->Ala161 or Glu161 are inactive in the induction of oocyte maturation. Here, we have analyzed the importance of an additional putative cdc2 phosphorylation site,Ser277. Single mutation of Ser277-->Asp277 or Ala277 had no effect on activity, and these mutants were unable to induce Xenopus oocyte maturation. However, the double mutant Ala161/Asp277 was capable of inducing oocyte maturation, suggesting that mutation of Ser277-->Asp277 could compensate for the mutation of Thr161-->Ala161. The Asp277 mutation could also compensate for the Ala161 mutation in the background of the activating mutations Ala14/Phe15. Although mutants containing the compensatory Ala161 and Asp277 mutations were capable of inducing oocyte maturation, these mutant cdc2 proteins lacked detectable in vitro kinase activity. Tryptic phosphopeptide mapping of mutant cdc2 protein and comparison with in vitro synthesized peptides indicated that Ser277 is not a major site of phosphorylation in Xenopus oocytes; however, we cannot rule out the possibility of phosphorylation at this site in a biologically active subpopulation of cdc2 molecules. The data presented here, together with prior reports of Ser277 phosphorylation in somatic cells, suggest an important role for Ser277 in the regulation of cdc2 activity. The regulatory role of Ser277 most likely involves its indirect effects on the nearby residue Arg275, which participates in a structurally important ion pair with Glu173, which lies in the same loop as Thr161 in the cdc2 protein.     

Mutational analysis of the interaction between active site residues and the loop region in mammalian purple acid phosphatases

Mammalian purple acid phosphatases (PAPs) can be divided into two groups, which exhibit distinct spectroscopic and kinetics properties: PAPs that consist of a single 36 kDa polypeptide, and PAPs that have undergone limited proteolysis to give two fragments with masses of 16 and 20 kDa, respectively. Proteolysis results in an increase in enzymatic activity, an increase in the optimal pH for activity, and a change in the g(z)() value of the characteristic EPR spectrum of the mixed-valence binuclear iron center. It has been proposed that these changes are due to the loss of interactions between Asp146 in an exposed loop region and active site residues upon proteolysis. In the present study, site-directed mutagenesis of Asp146 in recombinant rat bone PAP (recRPAP) has confirmed this hypothesis. Conversion of Asp146 into Ala, which eliminates the interaction of the side chain with the active site, resulted in an enzyme with properties typical of PAPs isolated in proteolytically cleaved forms. The Asp146Asn and Asp146Glu mutants were also prepared and examined to assess the effects of altered electrostatic interactions and side-chain length. Limited proteolysis of all three mutant enzymes with cathepsin L resulted in a significant increase in catalytic activity. Thus, although the interaction between Asp146 and (an) active site residue(s) is the major factor responsible for the low catalytic activity of uncleaved PAPs, other interactions are also important. Since both p-nitrophenyl phosphate and osteopontin, a potential in vivo substrate, show the same level of activation, the observed increase in catalytic activity upon proteolysis is likely to be due to electrostatic rather than steric effects. EPR spectra of FeZn-recRPAP before and after cleavage by cathepsin L suggest that cleavage primarily affects the divalent metal site. The observation that pK(es,1) is also sensitive to changes at the divalent site is consistent with the proposal that the nucleophilic hydroxide is that bridging the divalent and trivalent metals.     

Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts

Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly(143)-Gly(160) followed by the removal of a Val(161)-Ala(162) dipeptide at the N terminus of the C-terminal 16-kDa TRAP subunit. Cathepsin L initially released a shorter Gln(151)-Gly(160) peptide and completed processing at Ser(145) or Gly(143) at the C terminus of the N-terminal 23-kDa TRAP subunit and at Arg(163) at the N terminus of the C-terminal 16-kDa TRAP subunit. Mutation of Ser(145) to Ala partly mimicked the effect of proteolysis on catalytic activity, identifying Ser(145) as well as Asp(146) (Funhoff, E. G., Ljusberg, J., Wang, Y., Andersson, G., and Averill, B. A. (2001) Biochemistry 40, 11614-11622) as repressive amino acids of the loop region to maintain the TRAP enzyme in a catalytically latent state. The C-terminal sequence of TRAP isolated from rat bone was consistent with cathepsin K-mediated processing in vivo. Moreover, cathepsin K, but not cathepsin L, co-localized with TRAP in osteoclast-resorptive compartments, supporting a role for cathepsin K in the extracellular processing of monomeric TRAP in the resorption lacuna.     

Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2

Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.