A new l‐arginine oxidase engineered from l‐glutamate oxidase

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l‐Glutamate oxidase (LGOX) from Streptomyces sp. X‐119‐6 catalyzes the oxidative deamination of l‐glutamate to produce 2‐ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l‐glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l‐glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l‐arginine. The oxidative deamination activity of LGOX to l‐arginine is higher than that of l‐arginine oxidase form from Pseudomonas sp. TPU 7192. X‐ray crystal structure analysis revealed that the guanidino group of l‐arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild‐type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l‐arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l‐arginine by LGOX R305E, is proportional to the concentration of l‐arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l‐arginine. Thus, LGOX R305E is suitable for the determination of l‐arginine.     

Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein

RNase P is involved in processing the 5⿲ end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The K_m of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.     

The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues

The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid. Substitution of His(308) by arginine induced major changes in the donor substrate specificity of GlcAT-I. Interestingly, the H308R mutant was able to efficiently utilize nucleotide sugars UDP-glucose, UDP-mannose, and UDP-N-acetylglucosamine, which are not naturally accepted by the wild-type enzyme, as co-substrate in the transfer reaction. To gain insight into the role of Arg(277), site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg(277) with alanine abrogated the activity of GlcAT-I. Furthermore, the arginine-directed reagent 2,3-butanedione irreversibly inhibited GlcAT-I, which was effectively protected against inactivation by UDP-glucuronic acid but not by UDP-glucose. It is noteworthy that the activity of the H308R mutant toward UDP-glucose was unaffected by the arginine-directed reagent. Our results are consistent with crucial interactions between the His(308) and Arg(277) residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate.     

Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6

L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha2beta2gamma2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha2beta2gamma2 and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.     

Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.     

Contribution of basic residues of the autolysis loop to the substrate and inhibitor specificity of factor IXa

The autolysis loop (residues 143-154 in chymotrypsinogen numbering) plays a pivotal role in determining the macromolecular substrate and inhibitor specificity of coagulation proteases. This loop in factor IXa (FIXa) has 3 basic residues (Arg143, Lys147, and Arg150) whose contribution to the protease specificity of factor IXa has not been studied. Here, we substituted these residues individually with Ala in Gla-domainless forms of recombinant factor IX expressed in mammalian cells. All mutants exhibited normal amidolytic activities toward a FIXa-specific chromogenic substrate. However, Arg143 and Lys147 mutants showed a approximately 3- to 6-fold impairment in FX activation, whereas the Arg150 mutant activated factor X normally both in the absence and presence of factor VIIIa. By contrast, Arg143 and Lys147 mutants reacted normally with antithrombin (AT) in both the absence and presence of the cofactor, heparin. However, the reactivity of the Arg150 mutant with AT was impaired 6.6-fold in the absence of heparin and 33- to 70-fold in the presence of pentasaccharide and full-length heparins. These results suggest that Arg143 and Lys147 of the autolysis loop are recognition sites for FX independent of factor VIIIa, and Arg150 is a specific recognition site for AT that can effectively interact with AT only if the serpin is in the heparin-activated conformation.     

Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases

UDP-hexose 4-epimerases are critical in galactose metabolism and often important in lipopolysaccharide biosynthesis as well. Three groups of these enzymes have been reported based on their substrate specificity towards non-acetylated substrates (group 1), dual specificity towards N-acetylated and non-acetylated substrates (group 2) and specificity towards N-acetylated substrates (group 3). We recently reported the structure of a novel UDP-GalNAc 4-epimerase called WbgU and based on the structure proposed a model of specific substrate recognition by UDP-GalNAc 4-epimerases. In this work, we present an analysis of the proposed model of substrate recognition using site-directed mutagenesis of WbgU and crystal structure of the His305Ala mutant. This investigation reveals that the wild-type activity of WbgU is retained in most single-point mutants targeting the active site. However, a graded loss in activity is observed in double-and triple-point mutants with the quadruple-point mutant being completely inactive corroborating the proposed rationale of substrate recognition. Furthermore, crystal structure of the His305Ala mutant shows that the structure is significantly similar to the wild-type WbgU, albeit a loss in the critical hydrogen bond network seated at His305 and ensuing minor conformational changes. It is inferred that the specific and non-specific interactions throughout the active site confer it sufficient elasticity to sustain wild-type activity for several of the single-point mutations.     

Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase

Tyrosine phenol-lyase (TPL) and tryptophan indole-lyase (Trpase) catalyse the reversible hydrolytic cleavage of L-tyrosine or L-tryptophan to phenol or indole, respectively, and ammonium pyruvate. These enzymes are very similar in sequence and structure, but show strict specificity for their respective physiological substrates. We have mutated the active site residues of TPL (Thr(124), Arg(381), and Phe(448)) to those of Trpase and evaluated the effects of the mutations. Tyr(71) in Citrobacter freundii TPL, and Tyr(74) in E. coli Trpase, are essential for activity with both substrates. Mutation of Arg(381) of TPL to Ala, Ile, or Val (the corresponding residues in the active site of Trpase) results in a dramatic decrease in L-Tyr beta-elimination activity, with little effect on the activity of other substrates. Arg(381) may be the catalytic base with pK(a) of 8 seen in pH-dependent kinetic studies. T124D TPL has no measureable activity with L-Tyr or 3-F-L-Tyr as substrate, despite having high activity with SOPC. T124A TPL has very low but detectable activity, which is about 500-fold less than wild-type TPL, with L-Tyr and 3-F-L-Tyr. F448H TPL also has very low activity with L-Tyr. None of the mutant TPLs has any detectable activity with L-Trp as substrate. H463F Trpase also exhibits low activity with L-Trp, but retains high activity with other substrates. Thus, additional residues remote from the active site may be needed for substrate specificity. Both Trpase and TPL may react by a rare S(E)2-type mechanism.     

Site directed mutagenesis studies of FAD-dependent glucose dehydrogenase catalytic subunit of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

A FAD-dependent glucose dehydrogenase (FADGDH) mutant with narrow substrate specificity was constructed by site-directed mutagenesis. Several characteristics of FADGDH, such as high catalytic activity and high electron transfer ability, make this enzyme suitable for application to glucose sensors. However, for further applications, improvement of the broad substrate specificity is needed. In this paper, we mutated two residues, Asn475 and Ala472, which are located near the putative active site of the catalytic subunit of FADGDH and have been predicted from the alignment with the active site of glucose oxidase. Of the 38 mutants constructed, Ala472Phe and Asn475Asp were purified and their activities were analyzed. Both mutants showed a higher specificity toward glucose compared to the wild type enzyme.     

The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain.

Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.     

Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR

Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors. Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation. One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer. The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation. The findings imply complementarity between positively charged residues of the alpha2 helix of CheR and the negatively charged glutamates of the receptor. It is likely that this complementarity is involved in discriminating different methylation states of the receptors.     

Molecular basis for acceptor substrate specificity of the human beta1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively

The human beta1,3-glucuronosyltransferases galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) and galactose-beta1,3-glucuronosyltransferase P (GlcAT-P) are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective toward Galbeta1,3Gal (referred to as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand the molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information. The drastic effect of mutations of Glu227, Arg247, Asp252, and Glu281 on GlcAT-I activity indicated a key role for the hydrogen bond network formed by these four conserved residues in dictating Gal2 binding. Investigation of GlcAT-I determinants governing Gal1 recognition showed that Trp243 could not be replaced by its counterpart Phe in GlcAT-P. This result combined with molecular modeling provided evidence for the importance of stacking interactions with Trp at position 243 in the selectivity of GlcAT-I toward Galbeta1,3Gal. Mutation of Gln318 predicted to be hydrogen-bonded to 6-hydroxyl of Gal1 had little effect on GlcAT-I activity, reinforcing the role of Trp243 in Gal1 binding. Substitution of Phe245 in GlcAT-P by Ala selectively abolished Galbeta1,3Gal activity, also highlighting the importance of an aromatic residue at this position in defining the specificity of GlcAT-P. Finally, substituting Phe245, Val320, or Asn321 in GlcAT-P predicted to interact with N-acetylglucosamine (GlcNAc), by their counterpart in GlcAT-I, moderately affected the activity toward the reference substrate of GlcAT-P, N-acetyllactosamine, indicating that its active site tolerates amino acid substitutions, an observation that parallels its promiscuous substrate profile. Taken together, the data clearly define key residues governing the specificity of beta1,3-glucuronosyltransferases.     

Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity

Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analogue prodrugs in addition to its natural substrates, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine. To improve the low catalytic activity and tailor the substrate specificity of dCK, we have constructed libraries of mutant enzymes and tested them for thymidine kinase (tk) activity. Random mutagenesis was employed to probe for amino acid positions with an impact on substrate specificity throughout the entire enzyme structure, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. Kinetic analysis indicates that Arg104Gln/Asp133Gly creates a "generalist" kinase with broader specificity and elevated turnover for natural and prodrug substrates. In contrast, the substitutions of Arg104Met/Asp133Thr, obtained via site-saturation mutagenesis, yielded a mutant with reversed substrate specificity, elevating the specific constant for thymidine phosphorylation by over 1000-fold while eliminating activity for dC, dA, and dG under physiological conditions. The results illuminate the key contributions of these two amino acid positions to enzyme function by demonstrating their ability to moderate substrate specificity.     

Intra-subunit residue interactions from the protein surface to the active site of glutathione S-transferase AdGSTD3-3 impact on structure and enzyme properties

Structural residues are one of the major factors that modulate the catalytic specificity as well as having a role in stability of the glutathione S-transferases (GST). To understand how residues remote from the active site can affect enzymatic properties, four mutants, His144Ala, Val147Leu, Val147Ala and Arg96Ala, were generated. The selected residues appear to be in a putative intra-subunit interaction pathway from the exterior Asp150 to the active site Arg66 of AdGSTD3-3. The analysis of the four mutants suggested that the interaction formed between Asp150 and His144 is required for the packing of the hydrophobic core in domain 2. Mutations of both Asp150 and His144 impacted upon enzymatic properties. Two Val147 mutants also showed contribution to packing and support of the N-capping box motif by demonstrating shorter half-lives. The planar guanidinium of Arg96 is in a stacked geometry with the face of the aromatic ring of Phe140 in a cation-pi interaction. The Arg96 also interacts with several other residues one of which, Asp100, is in the active site. These interactions restrict movement of the residues in this region and as the data demonstrates when Arg96 is changed have dramatic impact on stability and enzyme properties. These findings indicate the significance of the roles played by residue interactions which can cause conformational changes and thereby influence the catalytic activity and stability of an enzyme.     

Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic toxin restrictocin

Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. Residues Tyr47, His49, Glu95, Phe96, Pro97, Arg120, and His136 have been predicted to form the active site of restrictocin. In this study, we have individually mutated these amino acids to alanine to probe their role in restrictocin structure and function. The role of Tyr47, His49, Arg120, and His136 was further investigated by making additional mutants. Mutating Arg120 or His136 to alanine or the other amino acids rendered the toxin completely inactive, whereas mutating Glu95 to alanine only partially inactivated the toxin. Mutation of Phe96 and Pro97 to Ala had no effect on the activity of restrictocin. The Tyr47 to alanine mutant was inactive in inhibiting protein synthesis, and had a nonspecific ribonuclease activity on 28S rRNA similar to that shown previously for the His49 to Ala mutant. Unlike the His136 to Ala mutant, the double mutants containing Tyr47 or His49 mutated to alanine along with His136 did not compete with restrictocin to cause a significant reduction in the extent of cleavage of 28S rRNA. In a model of restrictocin and a 29-mer RNA substrate complex, residues Tyr47, His49, Glu95, Arg120, and His136 were found to be near the cleavage site on RNA. It is proposed that in restrictocin Glu95 and His136 are directly involved in catalysis, Arg120 is involved in the stabilization of the enzyme-substrate complex, Tyr47 provides structural stability to the active site, and His49 determines the substrate specificity.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

Characterization of Escherichia coli uridine phosphorylase by single-site mutagenesis

The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results. In order to cast some light on the E. coli UP catalytic site, we mutagenized several residues in UP and measured by RP-HPLC the phosphorolytic activity of the mutant UP proteins in vitro. Mutations Thr94Ala, Phe162Ala, and Tyr195Gly caused a drastic decrease in UP activity. These three residues were suggested to be involved in the nucleoside binding site. However, surprisingly, Tyr195Ala caused a relative increase in enzymatic activity. Both Met197Ala and Met197Ser conserved low activity, suggesting a minor role for this residue in the UP active site. Glu196Ala completely lost UP activity, whereas the more conservative Glu196Asp mutation was still partially active, confirming the importance of maintaining the correct charge in the surroundings of this position. Glu198 was mutated to either Gly, Asp and Gln. All three substitutions caused complete loss of enzymatic activity suggesting an important role of Glu198 both in ribose binding and in interaction with phosphate ions. Arg30Ala and Arg91Ala eliminated UP activity, whereas Arg30Lys and Arg91Lys presented a very low activity, confirming that these residues might interact with and stabilize the phosphate ions. Ile69Ala did not decrease UP activity, whereas His8Ala lowered the activity to about 20%. Both amino acids were suggested to take part in subunit interactions. Our results confirm the structural similarity between E. coli UP and E. coli purine nucleoside phosphorylase (PNP).     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase

Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry.     

Ala657 and conserved active site residues promote fibroblast activation protein endopeptidase activity via distinct mechanisms of transition state stabilization

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.