Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540

The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from Serratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to family 20 of glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton donor and by the acetamido group of the substrate as a nucleophile. We investigated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemical characterization and by structural analyses of chitobiase -substrate co-crystals. We found that both residues are essential for chitobiase activity. The mutations, however, led to subtle changes in the catalytic site. Our results support the model that Glu540 acts as the proton donor and that Asp539 acts in several different ways. Asp539 restrains the acetamido group of the substrate in a specific orientation by forming a hydrogen bond with N2 of the non-reduced (-1) sugar. In addition, this residue participates in substrate binding. It is also required for the correct positioning of Glu540 and may provide additional negative charge at the active site. Thus, these biochemical and structural studies provide a molecular explanation for the functional importance and conservation of these residues.     

Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K(m) than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with approximately 2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K(m) than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.     

Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase.

Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (betaalpha)8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted -1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH-dependency of these effects, pKa calculations and available structural information. The results indicate that the pKa of the catalytic acid (Glu144) is 'cycled' during catalysis as a consequence of substrate-binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the -1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the -1 sugar and by increasing the pKa of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N-acetyl group of the -1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate.     

Mutational effects on transglycosylating activity of family 18 chitinases and construction of a hypertransglycosylating mutant

Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)(4) using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp(313/142) to Ala or the mutation of Asp(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromatic residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.     

Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans

The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.     

Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding

Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.     

Molecular dynamics simulations of a branched tetradecasaccharide substrate in the active site of a xyloglucan endo-transglycosylase

Molecular dynamics simulations of the tetradecasaccharide XXXGXXXG in complex with the hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 have been performed and analysed with respect to structure, dynamics, flexibility and ligand interactions. Notably, the charge state of the so-called ‘helper residue’ aspartate 87 (Asp87), which lies between the catalytic nucleophile [glutamate 85 (Glu85)] and general acid/base (Glu89) residues on the same beta strand, had a significant effect on PttXET16-34 active site structure. When Asp87 was deprotonated, electrostatic repulsion forced the nucleophile away from C1 of the sugar ring in subsite ? 1 and the proton–donating ability of Glu89 was also weakened due to the formation of a hydrogen bond with Asp87, whereas the protonation of Asp87 resulted in the formation of a hydrogen bond with the catalytic nucleophile and correct positioning of the catalytic machinery. The results suggest that catalysis in glycoside hydrolase family 16, and by extension clan GH-B enzymes, is optimal when the catalytic nucleophile is deprotonated for nucleophilic attack on the substrate, whereas the ‘helper residue’ and general acid/base residue are both in their conjugate acid forms to align the nucleophile and deliver a proton to the departing sugar, respectively.     

Snapping of the carboxyl terminal tail of the catalytic subunit of PKA onto its core: characterization of the sites by mutagenesis

A set of 45 mutants of the carboxyl terminal tail of the PKA catalytic subunit was prepared and used to assess the contribution of this tail to the structure and function of the kinase. Ala substitutions of Asp 323, Phe 327, Glu 333, and Phe 350 resulted in a complete loss of enzymatic activity. Other replacements by Ala (Phe 314, Tyr 330, Glu 332, and Phe 347) brought about either a drop in activity to less than 10% of the wild-type enzyme or a reduction of affinity toward ATP (Lys 317, Lys 319, Tyr 330, and Glu 332) or toward Kemptide (Ile 315, Tyr 330, Val 337, Ile 339, Lys 345, and Glu 346). Mutations of Ser 338, a major autophosphorylation site of PKA, by Ala, Glu, Asp, Gln, and Asn showed that the kinetic parameters of these mutants are similar to those of the wild-type. The contribution of each of these tail mutations to the structure and stability of the kinase was assessed by monitoring its effect on the heat stability (when measurable) or by determining the susceptibility of the mutant kinase to cleavage by the Kinase Splitting Membranal Proteinase/Meprin beta. Here we show that the tail of PKA has a key role in creating the active conformation of the kinase. It does so by means of specific amino acid residues, which act as "snapping points" to embrace the two lobes of the kinase and orient them in the correct juxtaposition for substrate docking, biorecognition, and catalysis.     

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.     

Contributions of long-range electrostatic interactions to 4-chlorobenzoyl-CoA dehalogenase catalysis: a combined theoretical and experimental study

It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-ligand complexes revealed an approximately 3-fold shift in the equilibrium of the two active site conformers away from that inducing strong pi-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.     

Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.     

Structure refinement of fructose-1,6-bisphosphatase and its fructose 2,6-bisphosphate complex at 2.8 A resolution

The structures of the native fructose-1,6-bisphosphatase (Fru-1,6-Pase), from pig kidney cortex, and its fructose 2,6-bisphosphate (Fru-2,6-P2) complexes have been refined to 2.8 A resolution to R-factors of 0.194 and 0.188, respectively. The root-mean-square deviations from the standard geometry are 0.021 A and 0.016 A for the bond length, and 4.4 degrees and 3.8 degrees for the bond angle. Four sites for Fru-2,6-P2 binding per tetramer have been identified by difference Fourier techniques. The Fru-2,6-P2 site has the shape of an oval cave about 10 A deep, and with other dimensions about 18 A by 12 A. The two Fru-2,6-P2 binding caves of the dimer in the crystallographically asymmetric unit sit next to one another and open in opposite directions. These two binding sites mutually exchange their Arg243 side-chains, indicating the potential for communication between the two sites. The beta, D-fructose 2,6-bisphosphate has been built into the density and refined well. The oxygen atoms of the 6-phosphate group of Fru-2,6-P2 interact with Arg243 from the adjacent monomer and the residues of Lys274, Asn212, Tyr264, Tyr215 and Tyr244 in the same monomer. The sugar ring primarily contacts with the backbone atoms from Gly246 to Met248, as well as the side-chain atoms, Asp121, Glu280 and Lys274. The 2-phosphate group interacts with the side-chain atoms of Ser124 and Lys274. A negatively charged pocket near the 2-phosphate group includes Asp118, Asp121 and Glu280, as well as Glu97 and Glu98. The 2-phosphate group showed a disordered binding perhaps because of the disturbance from the negatively charged pocket. In addition, Asn125 and Lys269 are located within a 5 A radius of Fru-2,6-P2. We argue that Fru-2,6-P2 binds to the active site of the enzyme on the basis of the following observations: (1) the structure similarity between Fru-2,6-P2 and the substrate; (2) sequence conservation of the residues directly interacting with Fru-2,6-P2 or located at the negatively charged pocket; (3) a divalent metal site next to the 2-phosphate group of Fru-2,6-P2; and (4) identification of some active site residues in our structure, e.g. tyrosine and Lys274, consistent with the results of the ultraviolet spectra and the chemical modification. The structures are described in detail including interactions of interchain surfaces, and the chemically modifiable residues are discussed on the basis of the refined structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase

The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8. Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.     

Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Kinetic analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has implicated a glutamate or aspartate residue in (i) formation of mevaldate thiohemiacetal by proton transfer to the carbonyl oxygen of mevaldate and (ii) enhanced ionization of CoASH by the resulting enzyme carboxylate anion, facilitating attack by CoAS- on the carbonyl carbon of mevaldate (Veloso, D., Cleland, W. W., and Porter, J. W. (1981) Biochemistry 81, 887-894). Although neither the identity of this acidic residue nor its location is known, the catalytic domains of 11 sequenced HMG-CoA reductases contain only 3 conserved acidic residues. For HMG-CoA reductase of Pseudomonas mevalonii, these residues are Glu52, Glu83, and Asp183. To identify the acidic residue that functions in catalysis, we generated mutants having alterations in these residues. The mutant proteins were expressed, purified, and characterized. Mutational alteration of residues Glu52 or Asp183 of P. mevalonii HMG-CoA reductase yielded enzymes with significant, but in some cases reduced, activity (Vmax = 100% Asp183----Ala, 65% Asp183----Asn, and 15% Glu52----Gln of wild-type activity, respectively). Although the activity of mutant enzymes Glu52----Gln and Asp183----Ala was undetectable under standard assay conditions, their Km values for substrates were 4-300-fold higher than those for wild-type enzyme. Km values for wild-type enzyme and for mutant enzymes Glu52----Gln and Asp183----Ala were, respectively: 0.41, 73, and 120 mM [R,S)-mevalonate); 0.080, 4.4, and 2.0 mM (coenzyme A); and 0.26, 4.4, and 1.0 mM (NAD+). By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition. During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme. By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support. Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme). Glutamate residue 83 of P. mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.     

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.     

Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae

Site-directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae. Kinetic parameters for 4-methylumbelliferyl-N,N'-diacetyl-chitobiose or 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the Km values were different. The function of the carboxyl group of Asp313 was partially replaced by the amide of Asn when the 4-methylumbelliferyl-N,N',N"-triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.     

Roles of Asp75, Asp78, and Glu83 of GTP-dependent phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis

The roles of Asp(75), Asp(78), and Glu(83) of the (75)DPSDVARVE(83) element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp(78) and Glu(83) are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp(78) influences Tyr(220); Tyr(220) helps to position bound PEP, and Glu(83) interacts with Arg(81). Experimental data on other PEPCKs indicate that Arg(81) binds PEP, and the phosphate of PEP interacts with Mn(2+) of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent K(m) value for Mn(2+) 170-fold. In contrast, Asp(75) is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, V(max) decreased 1.4-4-fold. K(m) values for PEP and Mn(2+) increased 3-9- and 1.2-10-fold, respectively. K(m) values for GDP and bicarbonate changed little. 2) For PEP formation, V(max) increased 1.5-2.7-fold. K(m) values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu(83)-Arg(81) interaction affected Arg(81)-PEP association. D78A change altered the Tyr(220)-PEP interaction. These events perturbed PEP-Mn(2+) interaction and consequently affected catalysis severely. In contrast, substitutions at Asp(75), a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp(75) substitutions affected PEP-Mn(2+) interaction by changing the positions of Asp(78), Arg(81), and Glu(83), which translated to differential effects on two directions.     

Soybean epoxide hydrolase: identification of the catalytic residues and probing of the reaction mechanism with secondary kinetic isotope effects

Soybean epoxide hydrolase catalyzes the oxirane ring opening of 9,10-epoxystearate via a two-step mechanism involving the formation of an alkylenzyme intermediate, which, in contrast to most epoxide hydrolases studied so far, was found to be the rate-limiting step. We have probed residues potentially involved in catalysis by site-directed mutagenesis. Mutation of His(320), a residue predicted from sequence analysis to belong to the catalytic triad of the enzyme, considerably slowed down the second half-reaction. This kinetic manipulation provoked an accumulation of the reaction intermediate, which could be trapped and characterized by electrospray ionization mass spectrometry. As expected, mutation of Asp(126) totally abolished the activity of the enzyme from its crucial function as nucleophile involved in the formation of the alkylenzyme. In line with its role as the partner of His(320) in the "charge relay system," mutation of Asp(285) dramatically reduced the rate of catalysis. However, the mutant D285L still exhibited a very low residual activity, which, by structural analysis and mutagenesis, has been tentatively attributed to Glu(195), another acidic residue of the active site. Our studies have also confirmed the fundamental role of the conserved Tyr(175) and Tyr(255) residues, which are believed to activate the oxirane ring. Finally, we have determined the secondary tritium kinetic isotope effects on the epoxide opening step of 9,10-epoxystearate. The large observed values, i.e. (T)(V/K(m)) approximately 1.30, can be interpreted by the occurrence of a very late transition state in which the epoxide bond is broken before the nucleophilic attack by Asp(126) takes place.     

Charged residues on a flap-loop structure of Lactococcus lactis prolidase play critical roles in allosteric behavior and substrate inhibition

Allosteric behavior and substrate inhibition are unique characteristics of Lactococcus lactis prolidase. We hypothesized that charged residues (Asp36, His38, Glu39, and Arg40), present on one loop essential for catalysis, interact with residues in or near the active site to impart these unique characteristics. Asp36 has a predominant role in the allosteric behavior, as demonstrated through the non-allosteric behavior of the D36S mutant enzyme. In contrast, a double mutant (D36E/R293K) maintained the allostery, indicating that this aspartic acid residue interacts with Arg293, previously shown to be critical in the allostery. Substitution of His38 drastically reduced the substrate inhibition, and substrate specificity of the mutant at Asp36 or His38 showed the influence of these residues to the substrate specificity. These findings confirm the importance of the loop in the enzymatic reaction mechanism and suggest the existence of conformational changes of the loop structure between open and closed states. A variety of mutations at Glu39 and Arg40 showed that these residues influence roles of the loop in the enzyme reaction. On the basis of these results and combined with observations of molecular models of this prolidase, we concluded that Asp36 and His38 interact with the residues in the active site to generate an allosteric subsite and a pseudo-S(1)' site, which are responsible for the allosteric behavior and substrate inhibition.     

Structure-guided mutational analysis of T4 RNA ligase 1

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.