Modulation of activity and substrate binding modes by mutation of single and double subsites +1/+2 and -5/-6 of barley alpha-amylase 1.

Enzymatic properties of barley alpha-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95-->Ala/Thr) and +1/+2 (Met298-->Ala/Asn/Ser) as well as in the double mutants, Cys95-->Ala/Met298-->Ala/Asn/Ser. Cys95-->Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95-->Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95-->Ala and wild-type. Met298-->Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are < or = 30% and < or = 10% of wild-type, respectively. The activity of Cys95-->Ala/Met298-->Ala/Asn/Ser is 100-180% towards starch, and the kcat/Km is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95-->Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6"'-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3.     

Preparation and enzymatic properties of subtilisin Novo chemically attached to soluble DEAE-dextran and insoluble DEAE-sephadex.

Analogous soluble and insoluble derivatives of subtilisin Novo (EC 3.4.21.14) were prepared by coupling the enzyme to CNBr-activated DEAE-dextran and DEAE-Sephadex, respectively. The DEAE-dextran-subtilisin displayed pH optima and Km values for ester hydrolysis similar to subtilisin, whereas the pH versus activity profiles obtained with DEAE-Sephadex-subtilisin were shifter towards the alkaline pH region and the Km values were increased. Compared with subtilisin, DEAE-dextran-subtilisin showed a 40-65% reduction of kcat for hydrolysis of N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester and benzyloxycarbonyl-glycyl-L-tyrosinamide and its maximum velocities for digestion of casein and clupein also amounted to 40-60% of the subtilisin values. With Deae-sephadex-subtilisin, in contrast, the maximum velocity of hydrolysis decreased to a greater extent for polypeptide substrates compared to ester substrates. The present results indicate that the chemical nature of a support can effect intrinsic properties of a matrix-bound enzyme in addition to the steric and diffusional effects usually observed with polymer-attached enzymes.     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

Construction and screening of a multi-point site-specific mutant library of subtilisin E with a set of oligonucleotides

A mutant library of subtilisin E containing random combinations of various mutagenized sites was constructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asn109Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.     

Construction and screening of a multi-point site- specific mutant library of subtilisin E with a set of oligonucleotides

A mutant library of subtilisin E containing random combinations of various mutagenized sites wasconstructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asnl09Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.     

Methionine residue accessibility in native subtilisin DY

The three methionine residues of subtilisin DY were specifically modified into methionine sulfoxide using increasing amounts of chloramine T. By means of subsequent treatment with cyanogen bromide, gel chromatography, Edman degradation of the obtained peptides and the known structure of subtilisin DY it was established that Met222 is exposed to the surrounding solution, Met124 is partially exposed and Met199 is buried. The data obtained were confirmed on a computer graphics space movable model of subtilisin Carlsberg where Met222 was seen to be on the surface of the molecule and Met199 shielded by Tyr262, Ala179 and Leu196. Upon oxidation of Met222 of subtilisin DY by chloramine T, 25% of its caseinolytic activity was lost. This can be explained by the immediate adjacency to the active-site Ser221. An additional 5% loss of activity was observed at each subsequent methionine modification.     

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of alpha-chymotrypsin and Carlsberg subtilisin with methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates

Methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates bearing different alkylthio groups were synthesized and employed as substrates for alpha-chymotrypsin and Carlsberg subtilisin in an attempt to investigate the properties of the hydrophobic pocket or cleft (S1 subsite) of the enzymes which accommodates the side-chain of the P1 amino acid residue of the substrates. The derivatives with ethylthio, 2-hydroxyethylthio, 2,3-dihydroxypropylthio, 2-aminoethylthio, carboxymethylthio, 2-carboxyethylthio, 1,2-dicarboxyethylthio, and 2-amino-2-carboxyethylthio (cysteinyl-S) groups were hydrolyzed by alpha-chymotrypsin but with kcat/Km values 4.6 to 15 times smaller than that of methyl N alpha-acetyl-L-tryptophanoate, due mainly to larger Km values. The glutathionyl derivative was only weakly bound to the enzyme. Analyses of the kinetic parameters suggested that the S1 pocket of alpha-chymotrypsin is rather more spacious than has been supposed and is able to interact flexibly with substrates so as to orient the scissile bond to the catalytic residues. On the other hand, none of the derivatives were hydrolyzed by Carlsberg subtilisin but they all inhibited the enzyme with Ki values which are generally smaller than the Km values for N alpha-acetyl-L-aromatic (modified aromatic) amino acid methyl esters. The S1 cleft of Carlsberg subtilisin interacts rather strongly with the derivatives but lacks the flexibility necessary for catalysis.     

Inactivation of Bacillus stearothermophilus Leucine Aminopeptidase II by Hydrogen Peroxide and Site-Directed Mutagenesis of Methionine Residues on the Enzyme

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.     

Functional analysis of CYP2D6.31 variant: homology modeling suggests possible disruption of redox partner interaction by Arg440His substitution

Cytochrome P450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous CYP2D6 alleles have been characterized at a functional level, although the consequences for expression and/or catalytic activity of a substantial number of rare variants remain to be investigated. One such allele, CYP2D6*31, is characterized by mutations encoding three amino acid substitutions: Arg296Cys, Arg440His and Ser486Thr. The identification of this allele in an individual with an apparent in vivo poor metabolizer phenotype prompted us to analyze the functional consequence of these substitutions on enzyme activity using yeast as a heterologous expression system. We demonstrated that the Arg440His substitution, alone or in combination with Arg296Cys and/or Ser486Thr, altered the respective kinetic parameters [Km (microM) and kcat (min(-1))] of debrisoquine 4-hydroxylation (wild-type, 25; 0.92; variants, 43-68; 0.05-0.11) and dextromethorphan O-demethylation (wild-type, 1; 4.72; variants, 12-23; 0.64-1.43), such that their specificity constants (kcat/Km) were decreased by more than 95% compared to those observed with the wild-type enzyme. The rates of oxidation of rac-metoprolol at single substrate concentrations of 40 and 400 microM were also markedly decreased by approximately 90% with each CYP2D6 variant containing the Arg440His substitution. These in vitro data confirm that the CYP2D6*31 allele encodes an enzyme with a severely impaired but residual catalytic activity and, furthermore, that the Arg440His exchange alone is the inactivating mutation. A homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 locates Arg440 on the proximal surface of the protein. Docking the structure of the FMN domain of human cytochrome P450 reductase to the CYP2D6 model suggests that Arg440 is a key member of a cluster of basic amino acid residues important for reductase binding.     

枯草蛋白酶E的突变体

Ser236位于横贯枯草蛋白酶E的α螺旋末端,远离催化活性中心,Ser236的突变不会对酶的活性产生大的影响。用定点突变的方法对枯草蛋白酶E的基因进行改造引入Ser236Cys,可能会形成分子间二硫键,有利于提高酶的稳定性。Ser236Cys变体酶(BP1)活性是野生型蛋白酶E的15倍,热稳定性提高3倍;进一步在其他位点引入突变的变体酶BU1(A1a15Asp/Gly20His/Ser236Cys)和BW1(Ser24His/Lys27Asp/Ser236Cys)活性都比野生型蛋白酶E低,但BW1的稳定性稍高于野生型蛋白酶E。     

Structural and functional roles of cysteine residues of Bacillus polymyxa beta-amylase.

Bacillus polymyxa beta-amylase contains three cysteine residues at positions 83, 91, and 323, which can react with sulfhydryl reagents. To determine the role of cysteine residues in the catalytic reaction, cysteine residues were mutated to construct four mutant enzymes, C83S, C91V, C323S, and C-free. Wild-type and mutant forms of the enzyme were expressed in, and purified to homogeneity from, Bacillus subtilis. A disulfide bond between Cys83 and Cys91 was identified by isolation of tryptic peptides bearing a fluorescent label, IAEDANS, from wild-type and C91 V enzymes followed by amino acid sequencing. Therefore, only Cys323 contains a free SH group. Replacement of cysteine residues with serine or valine residues resulted in a significant decrease in the kcat/Km value of the enzyme. C323S, containing no free SH group, however, retained a high specific activity, approximately 20% of the wild-type enzyme. None of the cysteine residues participate directly in the catalytic reaction.     

Thermal stable and oxidation-resistant variant of subtilisin E

A remarkable thermal stable and oxidation-resistant mutant was obtained using the random mutagenesis PCR technique on the mutant M222A gene of subtilisin E. Sequencing analysis revealed an A was replaced by G at nucleotide 671 of the subtilisin E gene, converting the asparagine codon (AAT) to serine codon (AGT) at position 118. The half-life of M222A/N118S enzyme activity, when heated at 65 degrees C, was approximately 80 min while the half-life of M222A and wild-type subtilisin E were 13 min and 15 min, respectively. This suggested the stability of the M222A/N118S mutant was five times greater than that of the wild-type enzyme. The mutant was also as oxidation resistant as the mutant M222A of subtilisin E. These results indicated the M222A/N118S mutant is both an oxidation-resistant and a heat-stable variant of subtilisin E.     

Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.     

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

Kinetic specificities of BPN' and Carlsberg subtilisins. Mapping the aromatic binding site.

The kinetic specificities of BPN' and Carlsberg subtilisins [EC 3.4.21.14] were examined with various nucleus-substituted derivatives of Nalpha-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of alpha-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two sutilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar trans-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2)-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5,500 +/- 700 s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.     

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.     

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active site of Escherichia coli alkaline phosphatase was investigated by site-specific mutagenesis. A mutant version of alkaline phosphatase was constructed with alanine in place of aspartic acid at position 101. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, pH 8.0, both the kcat and the Km for the mutant enzyme increase by approximately 2-fold, resulting in almost no change in the kcat/Km ratio. Under conditions of no external phosphate acceptor and pH 8.0, both the kcat and the Km for the mutant enzyme decrease by approximately 2-fold, again resulting in almost no change in the kcat/Km ratio. The kcat for the hydrolysis of 4-methyl-umbelliferyl phosphate and p-nitrophenyl phosphate are nearly identical for both the wild-type and mutant enzymes, as is the Ki for inorganic phosphate. The replacement of aspartic acid 101 by alanine does have a significant effect on the activity of the enzyme as a function of pH, especially in the presence of a phosphate acceptor. At pH 9.4 the mutant enzyme exhibits 3-fold higher activity than the wild-type. The mutant enzyme also exhibits a substantial decrease in thermal stability: it is half inactivated by treatment at 49 degrees C for 15 min compared to 71 degrees C for the wild-type enzyme. The data reported here suggest that this amino acid substitution alters the rates of steps after the formation of the phospho-enzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.