J. Biochem. 116, 176-182 (1994)

Role of the Conserved Glycyl Residues Located at the of Dehydrogenase from Bacillus stearothermophilus1

Toshihiro Sekimoto, Toshio Fukui, and Katsuyuki Tanizawa2 Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567

Received for publication, March 2, 1994

A tetrapeptide sequence, Gly-Gly-(Gly/Ala)-Lys, containing a catalytically important lysyl residue, is highly conserved in NAD(P)+-dependent amino acid dehydrogenases. To elucidate functional roles of the glycyl residues in this conserved sequence, Gly-77, Gly-78, and Gly-79 of the recombinant leucine dehydrogenase from Bacillus stearothermophilus have been individually replaced with Ala by site-directed mutagenesis. All of the mutant had Michaelis constants for ƒ¿-keto-iso-caproate and ammonia several times larger than the wild-type while retaining considerable catalytic activities. However, inhibition constants for a analog without an ƒ¿-carbonyl group were unchanged by the mutations. On the other hand, the rate of inactivation by pyridoxal 5•L-phosphate and the microenvironment of aromatic residues , in particular of the sole tryptophanyl residue (Trp-46) located in the vicinity of the active site, were affected by the mutations of the glycyl residues. All of these results suggest that the conserved glycyl residues are important for fine-tuning of the position and/or orientation of the ƒÃ-amino group of Lys-80 at the active site to function efficiently as a general-base catalyst. Furthermore, the Gly-77 and Gly-78 mutant enzymes had markedly decreased thermal stabilities, showing that these two glycyl residues are also critical for the conformational stability of this thermostable enzyme.

Key words: active site, glycine-rich sequence, leucine dehydrogenase, site-directed mutagenesis, thermal stability.

Leucine dehydrogenase [EC 1.4.1.9], which catalyzes the site-directed mutagenesis and pH analysis for the steady- reversible deamination of L-leucine and some other branch- state kinetic parameters (17). ed-chain L-amino acids to the corresponding keto acids, Further comparison of the local sequence around the belongs to a superfamily of NAD(P)+-linked amino acid active-site Lys-80 of leucine dehydrogenase with those of dehydrogenases. The enzyme cloned from Bacillus stearo other amino acid dehydrogenases has revealed that a thermophilus (1) shares considerable sequence similarities glycine-rich tetrapeptide sequence, Gly-Gly-(Gly/Ala)- in the catalytic and coenzyme-binding domains with the Lys, including the catalytic lysine, is highly conserved (1- enzymes acting on other amino acids, including glutamate 13). However, the significance of the conservation and the (2-11), phenylalanine (12, 13), and alanine (14) dehydro functional role(s) of these glycyl residues in the conserved genases, although the overall similarities among these sequence are unknown. X-ray crystallographic study of enzymes are not high, except for the homology of about 50% from Clostridium symbiosum, between leucine and phenylalanine dehydrogenases. We solved to 1.9 •ð resolution, has indicated that the two previously identified an active-site lysine (Lys-80) in the conserved glycyl residues (Gly-122 and Gly-123, equiva recombinant leucine dehydrogenase by modification with lent to Gly-77 and Gly-78, respectively, of leucine dehy PLP (15). This lysyl residue is totally conserved in the drogenase) are situated close together in a pocket adjacent catalytic domains of those amino acid dehydrogenases and to the B-face of the nicotinamide ring of the bound has been shown to bear a general-base function in the (18, 19). To elucidate the functional role(s) of the con by our recent site-directed mutagenesis studies served glycyl residues of leucine dehydrogenase from B.

(16). Another active-site lysyl residue (Lys-68), which is stearothermophilus, the three glycyl residues (Gly-77, involved, in its protonated form, in binding of the ƒ¿-car Gly-78, and Gly-79) have been individually replaced by boxyl group of substrate, has also been identified by alanine via site-directed mutagenesis. The results de scribed in this paper suggest that these glycyl residues of 1 This study was supported by a grant from the Osaka University leucine dehydrogenase are important for optimizing the

Institute of Scientific and Industrial Research/Welding Research position and/or orientation of the e-amino group of Lys-80 Institute joint research program, "Advanced Materials Creation and to act efficiently as the general-base catalyst. In addition, Their Limit State Prediction for Environment Preservation." Gly-77 and Gly-78 also play a critical role in the confor 2 To whom correspondence should be addressed . Abbreviations: G77A, the mutant enzyme of leucine dehydrogenase, mational stability of this thermostable enzyme. in which Gly-77 is replaced by Ala (other mutant enzymes are abbreviated in the same manner); PLP, pyridoxal 5•L-phosphate.

176 J. Biochem. Role of Conserved Glycines in Leucine Dehydrogenase 177

equal volumes of 0.2 M 2. (N-morpholino)ethanesulfonic EXPERIMENTALPROCEDURES acid (MES), NN-bis(2-hydroxyethyl)glycine (Bicine), and cyclohexylaminopropanesulfonic acid (CAPS), and titrat- Site-Directed Mutagenesis-Substitutions of Ala for ing to the desired pH values with NaOH. The ionic Gly-77, Gly-78, and Gly-79 , and of Phe for Trp-46 of strengths of buffers were maintained at 0.1 by addition of leucine dehydrogenase were performed by the method of an appropriate amount of NaCl calculated according to Ellis Kunkel et al. (20), as described previously (16) . The and Morrison (22). The variations with pH of the values for following four oligonucleotide primers were synthesized V/K were analyzed by fitting the data to Eq. 1 using the with an Applied Biosystems DNA synthesizer model 381 to program BELL (20), contain appropriate mismatched bases (indicated by aster- isks) in the complementary codons for Gly-77 , Gly-78, Gl y-79, and Trp-46 (underlined): where y represents the value of V/K at a particular pH, C G77A: 5'-TTGCCCCCGGCCAAGTTGA-3' is the pH-independent value of the parameter, and K,, and Kb are acid dissociation constants associated with ionizable G78A: 5'-CGTTTTGCCCGCGCCCAAGTT-3' groups on the acid limb and alkaline limb, respectively, of G79A: 5'-ACCGTTTTGGCCCCGCCC-3' pH profiles. Modification with PLP The purified wild-type and W 46F : 5' -AATTGTACAAAACATGCGCGTCC-3' three Gly mutant enzymes (10 j M subunit) were incubated at 30'C with 1 mM PLP in 50 mM HEPES buffer (pH 7.5) After confirming the nucleotide sequence 1-666 corre- in a final volume of 0.1 ml. At various time intervals (1-15 sponding to the region from the translational initiation min), 10-,ul aliquots of the reaction mixture were with- codon ATG to the Sail site in e the mutant gene obtained, the drawn, freshly prepared solution of NaBH4 was added to a 1.1-kbp HindIII-Sail fragment containing the mutated site final concentration of 20 mM to stop the reaction, and was excised from the replicative form M13 mp19 phage residual activities were assayed, as described previously DNA, ligated into the HindIII-SaII site of pBLeuDH (15), (15). To determine the labeled sites, the enzyme (5 nmol and transformed into E. coli JM109 cells. The wild-type, subunit) modified with 1 mM PLP for 4 min and reduced G79A, and W46F mutant enzymes were purified to homo- with NaBH4 was lyophilized, suspended in 0.1 ml of 0.1 M geneity from the crude extracts of the recombinant E. coli ammonium bicarbonate buffer (pH 8.0) containing 4 M cells grown at 37TC for 12 h in 1 liter of Luria broth (1.0% urea, and then diluted with 0.1 ml of 0.1 M ammonium tryptone, 0.5% NaCl, and 0.5% yeast extract) containing 50 bicarbonate. N-Tosyl-L-phenylalanine chloromethyl- ,ug/ml sodium ampicillin and 0.5 mM isopropyl-l-thio-,8- ketone-treated trypsin (Millipore) was added to the turbid D-galactopyranoside, as described previously (15). Because solution in a 1 : 50 (mol/mol) ratio of protease to substrate. the G77A and G78A mutant enzymes showed very low Digestion was performed at 37'C for 24 h, and the peptides thermal stability compared with the wild-type enzyme (see produced were separated by HPLC under the conditions below), both of the mutant enzymes were purified without described previously (15). heat treatment (at 70'C for 30 min). CD Measurements-CD spectra were measured at 25'C Steady-State Kinetic Analysis-The oxidative deamina- in 10 mM potassium phosphate buffer, pH 7.2, with a Jasco tion of L-leucine and the reductive amination of a -keto- iso- spectropolarimeter model J-600. In the calculation of the caproate were measured by monitoring spectrophoto- mean residue ellipticity (0), the mean residue weight was metrically the appearance and disappearance, respectively, taken to be 111 for the enzyme protein. The CD spectra of NADH under the conditions described previously (16). were obtained at a protein concentration of 0.2 mg/ml in a The steady-state kinetic parameters were determined by 2.0-cm light path length cuvette for measurements in the varying systematically the concentrations of both substrate wavelength region above 250 nm and at a protein concen- and coenzyme, except for ammonia, which was held at a tration of 0.1 mg/ml in a 0.1-cm light path length cuvette constant, saturating concentration (1.0 M), and NADH (0.1 for measurements in the wavelength region below 250 nm. mM). In determination of inhibition constants for iso- Fluorescence Measurement-Fluorescence emission caproate, concentrations of both substrate and inhibitor spectra of the wild-type and mutant enzymes (1,u M) were were varied. Kinetic constants were calculated from the measured at 25'C in 50 mM potassium phosphate buffer initial rates by the nonlinear regression method using (pH 7.2) with a Hitachi spectrofluorometer model F-4010. FORTRAN programs (HYPER, SEQUEN, and COMP) The fluorescence emission due to Trp-46 was recorded upon described by Cleland (21). Maximal activity (V) was excitation at 295 nm. expressed as turnover numbers (s-1) using a calculated Thermal Stability-Thermal stabilities of the wild-type, molecular weight of 46,900 per subunit (1), each having one G77A, G78A, and G79A mutant enzymes were analyzed by active site. monitoring the protein unfolding process by heat following pH Studies-Prior to the analysis of pH dependence of the changes in ellipticity at 222 nm. All measurements kinetic constants, stabilities of the wild-type and three Gly were carried out in 10 mM potassium phosphate buffer, pH mutant enzymes in buffers of various pH values (pH 7.2- 7.2, at a protein concentration of 0.5 mg/ml. The tempera- 10.8) were confirmed by measuring the remaining activity ture was raised at a constant rate of 1'C/min through a after incubation at 25'C for 30 min; only the G78A mutant thermostatic cell holder and was measured directly in the enzyme showed slightly reduced pH stability above pH cuvette with a Rikagaku Kogyo thermometer model DP- 10.2. For determination of V/K values for L-leucine in the 500. oxidative deamination, buffers were prepared by mixing

Vol. 116, No. 1, 1994 178 T. Sekimoto et al.

previously observed similarly increased Km values for ƒ¿-keto-iso-caproate of the K80A and K80Q mutant en RESULTS zymes of leucine dehydrogenase (16), in which the ƒÃ-amino

Purification of Leucine Dehydrogenase Mutated at Gly- group of Lys-80, interacting with the ƒ¿-carbonyl group of 77, Gly-78, and Gly-79•\To examine whether the three ƒ¿-keto -iso-caproate , is missing. conserved glycyl residues of leucine dehydrogenase from B. Inhibition by Iso-Caproate•\The observation that all stearothermophilus have any functional role(s), they were three Gly mutant enzymes show specific increases in the Km replaced individually with Ala by site-directed mutagene values for ƒ¿-keto-iso-caproate suggested that the interac sis as described under "EXPERIMENTAL PROCEDURES." The tion between the ƒÃ-amino group of the catalytic Lys-80 and G79A mutant enzyme could be purified to homogeneity by the ƒ¿-carbonyl group of the keto acid substrate was the same procedure as the wild-type enzyme, whereas the perturbed in these mutant enzymes. To substantiate this, G77A and G78A mutant enzymes were purified by only a we analyzed the inhibition in the reductive amination of the single step of ion-exchange chromatography (DEAE-Toyo wild-type and three Gly mutant enzymes by iso-caproate, a pearl) without heat treatment, because they had very low substrate analog without an ƒ¿-carbonyl group, and found thermal stabilities compared with the wild-type enzyme that it inhibits the wild-type and mutant enzymes competi (see below). All the wild-type and mutant enzymes were tively with ƒ¿-keto-iso-caproate. Contrary to the marked >95% homogeneous as judged by SDS-PAGE (data not difference in Km values for ƒ¿-keto-iso-caproate, the K1 shown). values for iso-caproate were almost the same for the Steady-State Kinetic Analysis-According to the ordered wild-type and three Gly mutant enzymes (Table I). This Bi-Ter mechanism, in which NAD+ and L-leucine are result is compatible with the interpretation that the inter- bound, and NH4+, ƒ¿-keto-iso-caproate, and NADH are action between Lys-80 and the keto acid substrate was released in that order (16), steady-state kinetic parameters affected unfavorably in the Gly mutant enzymes, probably of the wild-type and three mutant enzymes were deter because the position and/or orientation of the ƒÃ-amino mined as summarized in Table I. In the oxidative deamina group of Lys-80 at the active site was changed, although tion, the kmax values of the G77A, G78A, and G79A mutant slightly, by the mutations of these glycyl residues. enzymes were 36, 5.4, and 40%, respectively, of that of the pH Dependence of Kinetic Parameters-The wild-type wild-type enzyme. In the reductive amination, the kmax enzyme shows a bell-shaped pH profile of log(V/K) for values of the three mutant enzymes were comparable to L-leucine including two ionizable groups with pKa=8.9•} that of the wild-type enzyme. Such significantly high activities of mutant enzymes of leucine dehydrogenase in the reductive amination as compared with those in the oxidative deamination were observed previously with the K80A and K68A mutant enzymes, and were ascribed to stimulation by ammonia, one of the substrates in the amination reaction (16, 17). Due to this stimulation, the activities of the K80A and K68A mutant enzymes in the oxidative deamination were markedly enhanced by ex ogenously added ammonia (16, 17). Therefore, we ex amined the effect of ammonia on the activities of the three Gly mutant enzymes in the oxidative deamination, but none of the Gly mutant enzymes was activated by exogenously added ammonia (up to 1M, data not shown). As for Km values for NAD+ and NADH, replacements of the conserved glycyl residues by Ala caused essentially no change, suggesting that these three glycyl resides are not involved in binding of the nicotinamide coenzyme. In contrast, all of the three Gly mutant enzymes showed significant changes in Km values for substrates in the reductive amination. Compared with the wild-type en Fig. 1. pH Dependences of steady-state kinetic parameters in zyme, the G77A, G78A, and G79A mutant enzymes respec the oxidative deamination. V/K values for L-leucine (M-1•Es-1) of tively showed higher values for ƒ¿-keto-iso-caproate by the wild-type enzyme (•œ), the G77A mutant enzyme (•›), the G78A 6.3-, 8.8-, and 6.4-fold, and for ammonia by 2.8-, 10-, and mutant enzyme (•¢), and the G79A mutant enzyme (•£) were plotted 3.9-fold. The Km values for L-leucine were essentially the against pH. The curves are a nonlinear least-squares best fit of the same for the wild-type and three Gly mutant enzymes. We experimental data.

TABLE I. Steady-state kinetic parameters of the wild-type and mutant enzymes of leucine dehydrogenase.

J. Biochem. Role of Conserved Glycines in Leucine Dehydrogenase 179

Fig. 2. Inactivation of the wild-type and mutant en zymes of leucine dehydrogen ase by PLP. The enzymes (10ƒÊ M subunit) were incubated at 30•Ž with 1mM PLP in 50mM HEPES buffer (pH 7.5). At each indicated time, aliquots were withdrawn and reduced with 20 mM NaBH4, and the remaining activities were assayed for the wild-type enzyme (•œ), the G77A mutant enzyme (•›), the G78A mutant enzyme (•£), and the G79A mutant enzyme (•¢) in (A). In (B), the enzymes (5nmol subunit) incubated with 1mM PLP for 4min and reduced with NaBH4 were digested with trypsin as described under "EXPERIMENTAL PROCE

DURES," and the digests were chromatographed on a C18 col umn (15). The peptides were detected by continuous monitoring of the absorbance at 215nm (not shown) and the fluorescence (excitation at 330nm and emission at 395nm). The highest fluorescent peak corresponds to the peptide containing the PLP-labeled Lys-80 (15).

the wild-type enzyme. For the G78A mutant enzyme, the pKa values of both Lys-80 (pKa=9.4•}0.1) and Lys-68 (pKa=10.1•}0.1) were considerably altered by the muta tion. Inactivation by PLP-We previously demonstrated that leucine dehydrogenase from B. stearothermophilus is inac tivated by incubation with PLP as a result of the modifica tion of the catalytically important Lys-80 (15). Since the

pKa values of Lys-80 have been changed in the three Gly mutant enzymes as described above, its reactivity toward PLP might also be affected by the mutations of the glycyl residues. Thus the inactivation by PLP of the three Gly mutant enzymes was examined (Fig. 2A). The inactivation rates of the G78A and G79A mutant enzymes were faster than that of the wild-type enzyme, but that of the G77A mutant enzyme was almost the same as that of the Fig. 3. CD spectra of the wild-type and mutant enzymes of wild-type enzyme. The HPLC profiles for the tryptic leucine dehydrogenase. The CD spectra were measured at a protein digests of the wild-type and mutant enzymes modified with concentration of 0.2mg/ml in the region above 250nm and at 0.1mg/ ml in the region below 250nm. The unit of [ƒÆ] is mdeg•Ecm2•Edmol-1. PLP for a short period (4 min) revealed that the label was incorporated almost exclusively into the tryptic peptide containing Lys-80 (15) in the wild-type, G77A, and G79A mutant enzymes (Fig. 2B). It is obvious, however, that PLP 0.1 and 10.7•}0.1 (16). The low pKa group on the acid limb was incorporated into several peptides other than that has been identified as the ƒÃ-amino group of Lys-80, which containing Lys-80 in the G78A mutant enzyme, the one plays a role as the general-base catalyst in the reaction that was most rapidly inactivated (Fig. 2A), suggesting that (16), and the high pKa group on the alkaline limb has been other active-site lysyl residues such as Lys-68 (17) might ascribed to the ƒÃ-amino group of Lys- 68, which interacts, in its protonated form, with the ƒ¿-carboxyl group of sub be modified with PLP to inactivate this enzyme more rapidly than the wild-type and the other two mutant strates (17). Since it seems reasonable to assume that the enzymes. In addition, the fluorescence peak due to the pKa values of ionizable groups located at the active site are Lys-80-containing peptide of the G79A mutant enzyme is sensitive to conformational changes of the active site, the higher by about 50% than that of the wild-type enzyme pH dependences of steady-state kinetic parameters for the oxidative deamination of L-leucine catalyzed by the three (Fig. 2B). These results show that the reactivity toward PLP of the ƒÃ-amino group of Lys-80 (and also other Gly mutant enzymes were also analyzed (Fig. 1). All three active-site lysyl residues in the G78A mutant enzyme) mutant enzymes, like the wild-type enzyme, showed bell- have been affected by the mutations into Ala of Gly-78 and shaped pH profiles of log(V/K) for L-leucine. With the Gly-79, even though their changes in pKa values of Lys-80 G77A and G79A mutant enzymes, the pKa values of Lys-80 do not correlate directly with the reactivities toward PLP. shifted toward the acidic side (pKa=8.5•}0.1 for G77A and CD Spectra-CD spectra of the wild-type and three Gly pKa=8,7•}0.1 for G79A, calculated according to Eq. 1), mutant enzymes were measured to see whether global whereas the pKa values of Lys-68 were almost the same as

Vol. 116, No. 1, 1994 180 T . Sekimoto et al.

TABLE II. Thermal stability of the wild-type and mutant enzymes of leucine dehydrogenase. Tm is the temperature at the midpoint of the unfolding transition by heat. The thermal transition curve was analyzed by assuming the two-state approximation and the equilibrium constant of unfolding (KD=N/D) was determined from the equation KD=fD/(1-fD), where fD is the fraction of the unfolded moleculleeat each temperature. The change in the melting tempera ture, ĢTm, of each mutant enzyme was the difference from the Tm value of the wild-type enzyme. The change in the free energy of unfolding of the mutant enzymes relative to that of the wild-type enzyme, ĢĢG, was calculated from ĢĢG=ĢTmĢS (24), where ĢS is the unfolding entropy change of the wild-type enzyme determined from the vent Hoff plot.

enzyme exhibited only weak fluorescence emission around 310nm, which was probably attributable to tyrosyl resi Fig. 4. Fluorescence emission spectra of the wild-type and mutant enzymes of leucine dehydrogenase. The excitation wave- dues excited slightly at 295 nm. When the wild-type length was 295nm, and protein concentration was 1ƒÊM in 50mM enzyme was denatured in the presence of 6M guanidine potassium phosphate buffer (pH 7.2). The emission spectra of the hydrochloride, the fluorescence intensity decreased to wild-type enzyme (•œ), the W46F mutant enzyme (•¡), and the about half of that in the native state, showing that the wild-type enzyme in the presence of 6M guanidine hydrochloride (•›) tryptophanyl residue is fully exposed to solvent by denatu were recorded in (A). The spectra of the wild-type enzyme (•œ), the ration (Fig. 4A). However, a red shift of the fluorescence G77A mutant enzyme (•›), the G78A mutant enzyme (•£), and the G79A mutant enzyme (•¢) were recorded in (B). peak, which is often observed when tryptophans in a hydrophobic environment are exposed to solvent by de naturation of the protein, did not take place. It is, therefore, conformational changes were brought about by the replace suggested that Trp-46 of leucine dehydrogenase may be in ments of Gly-77, Gly-78, and Gly-79 by Ala. All the mutant a solvent-accessible environment to some extent. enzymes showed CD spectra practically identical with that The florescence emission spectrum of the G79A mutant of the wild-type enzyme in the 200-250nm region (Fig. 3), enzyme overlapped with that of the wild-type enzyme, indicating that the wild-type and mutant enzymes contain while those of the G77A and G78A mutant enzymes very similar secondary structures. On the other hand, the blue-shifted about 5 and 10nm, respectively, as compared spectra of the Gly mutant enzymes in the 260-290nm with that of the wild-type enzyme (Fig. 4B). This indicates region, which reflects the environment of aromatic resi that the environment around Trp-46 of these two mutant dues, were different from that of the wild-type enzyme enzymes became more hydrophobic than that of the (Fig. 3), showing that the mutations of these Gly residues wild-type enzyme, and the extents of the fluorescence shifts had caused local conformational changes around some are consistent with the decreases in the near-UV CD bands aromatic residues. To estimate which aromatic residue(s) of the two mutant enzymes, as described above. contribute most prominently to the CD band in the 260-290 Thermal Stability and Susceptibility to Limited Proteoly nun region, the sole tryptophanyl residue (Trp-46) in sis•\Although the overall conformation of the enzyme leucine dehydrogenase from B. stearothermophilus (1) was protein appeared to be unchanged by the mutations of the mutated into Phe as described under "EXPERIMENTAL three glycyl residues as judged from the far-UV CD spectra PROCEDURES," and the CD spectrum of the purified W46F of the mutant enzymes (see above), we noticed during mutant enzyme was measured (Fig. 3). The W46F mutant purification that the G77A and G78A mutant enzymes enzyme showed only a very weak CD band in the 260-290 completely lost their activities upon heat treatment at 70•Ž nm region, comparable to that of the G78A mutant enzyme, for 30min. This was probably due to their lowered thermal indicating that the CD band of the wild-type enzyme in this stabilities. Assuming the two-state unfolding approxima region is mainly due to Trp-46. Thus the mutations of the tion, the thermal transition process was measured by the three glycyl residues might affect to various extents the changes in ellipticity at 222nm by heat treatment of the local conformation around Trp-46, which is predicted to be wild-type and three Gly mutant enzymes, and thermody located in the vicinity of the active site on the basis of the namic parameters for the unfolding process were calculated X-ray crystallographic structure of the synonymous gluta according to the method of Pace and Laurents (23). Table mate dehydrogenase from Clostridium symbiosum (18, 19). II summarizes the unfolding temperatures (Tm) of the Fluorescence of Trp-46•\The changes in the microen wild-type and mutant enzymes, and the inferred changes in vironment of Trp-46 were further examined by measuring the free energy of unfolding relative to that of the wild-type the fluorescence emission spectra of the wild-type and three enzyme (ƒ¢ƒ¢G). All the mutant enzymes showed lowered Gly mutant enzymes. The wild-type enzyme emitted Tm values compared with the wild-type enzyme. Strikingly, significant fluorescence around 355 nm upon excitation at the G77A and G78A mutant enzymes were much less 295nm (Fig. 4A). The fluorescence observed was confirmed stable, being unfolded at temperatures lower by 19.5 and to be due solely to Trp-46, because the W46F mutant 26.5•Ž, respectively, than the wild-type enzyme.

J. Biochem. Role of Conserved Glycines in Leucine Dehydrogenase 181

To further examine the conformational stability of the contribution of Lys-80 in the oxidative deamination than in wild-type and mutant enzymes, susceptibility to digestion the reductive amination (16) may be one reason. by protease was also examined under non-denaturing All three Gly mutant enzymes showed Km values for conditions by incubating the enzymes at 37•Ž for 15h with ƒ¿-keto-iso-caproate and ammonia several times larger trypsin or subtilisin at a protease-to-substrate ratio of 1: than those of the wild-type enzyme (Table I). We previous- 25 (mol/mol). When analyzed by the remaining activities ly observed similarly increased Km values for ƒ¿-keto-iso- and SDS-PAGE (data not shown), the wild-type and G79A caproate with the K80A and K80Q mutant enzymes of mutant enzymes were found to be digested at almost the leucine dehydrogenase, in which the ƒÃ-amino group of same rate, whereas the degradation rates of the G78A and Lys-80 interacting with the ƒ¿-carbonyl group of ƒ¿-keto- G77A mutant enzymes were faster than that of the iso-caproate in the amination reaction is absent (16). On wild-type enzyme in this order. the other hand, the K, values for iso-caproate without an ƒ¿-carbonyl group were almost the same for the wild-type DISCUSSION and three Gly mutant enzymes (Table I). These findings suggest that the increases in Km values of all the three Gly A sequence motif of Gly-Gly-(Gly/Ala)-Lys including the mutant enzymes for ƒ¿-keto-iso-caproate and probably also catalytically important lysyl residue is highly conserved in for ammonia are caused by an indirect effect on the interac most of the amino acid dehydrogenases sequenced to date tion between the ƒÃ-amino group of Lys-80 and the ƒ¿- (1-14). Similar motifs containing several glycyl residues carbonyl group of the bound substrate. From the pH close to a reactive lysyl residue have been found in nucleo dependence of log(V/K) for L-leucine, the pKa values of tide-binding proteins (25-27). However, the functional the e-amino group of Lys-80 in the three mutant enzymes roles of the conserved glycyl residues in these glycine-rich were found to be changed, although slightly, and the rates sequences are not fully understood. One possibility is that of inactivation by PLP were also affected in the G78A and the conserved glycyl residues provide flexibility to the G79A mutant enzymes. Dihedral angles of a glycyl residue region, which frequently forms a loop structure, as suggest- can be within a wide range due to the absence of a bulky side ed for the porcine muscle adenylate kinase (28) and E. coli chain, and replacement of Gly by Ala would restrict the glycogen synthase (29). Another possibility is that the angles. This is probably the main reason for changing glycyl residues themselves interact directly with substrate, conformations of active-site residues. It is therefore sug as observed in triose phosphate (30). The X-ray gested that the mutations of the three conserved glycyl crystallographic study of glutamate dehydrogenase from C. residues affect the position and/or orientation of the symbiosum complexed with the substrate L-glutamate (19) ƒÃ-amino group of Lys-80 at the active site and that these showed that the conserved glycyl residues (Gly-122 and glycyl residues, particularly Gly-78, are important for Gly-123, equivalent to Gly-77 and Gly-78, respectively, of fine-tuning of the optimal conformation of the ƒÃ-amino leucine dehydrogenase) form a part of the active site and group of Lys-80 to function efficiently as a general-base are situated in a pocket close to the B-face of the nicotin catalyst. Based on the largest effects caused by the amide ring of the bound cofactor, but not involved directly mutation of Gly-78 on the near-UV CD spectrum, the in substrate binding; they have structural rather than fluorescence peak position due to the nearby Trp-46, the functional roles. rate of inactivation by PLP, and the catalytic activity in the We recently provided evidence that Lys-80 of leucine reductive amination, the perturbation of the active-site dehydrogenase participates in the catalysis as a general conformation may be most prominent with the G78A base, assisting the nucleophilic attack of a water molecule mutant enzyme. The proposed role of the conserved glycyl against the substrate ƒ¿-carbon atom (16). The three Gly residues is probably common to glutamate and phenylala mutant enzymes prepared in this study, in which each nine dehydrogenases, both of which share considerable conserved glycyl residue close to this Lys-80 is replaced by similarity in the active-site structure with leucine dehy Ala, a residue minimally different from Gly in the side- drogenase (31). chain bulkiness, exhibited catalytic activities comparable Of the three glycyl residues in the conserved tetrapep to those of the wild-type enzyme (Table I). This indicates tide sequence of leucine dehydrogenase, the residue corre that neither Gly-77, Gly-78, nor Gly-79 directly partici sponding to Gly-79 is substituted by Ala in glutamate dehydrogenase from bovine and human livers (3, 6), and pates in the catalysis. Only the G78A mutant enzyme had 5.4% of the wild-type enzyme activity in the oxidative the residue corresponding to Gly-77 is substituted by His in deamination. Such marked differences between the activ alanine dehydrogenase from B. stearothermophilus and B. ities of oxidative deamination and reductive amination sphaericus (14). The lower conservation of those two were observed previously for the K80A and K68A mutant residues is consistent with their having less functional enzymes of leucine dehydrogenase (16, 17), and these importance than Gly-78. The wild-type leucine dehydrogenase from B. stearother phenomena were ascribed to stimulation by ammonia, one of the substrates in the reductive amination, taking the mophilus shows high thermal stability and its Tm value in the heat denaturation has been determined to be about 80•Ž place of the ƒ¿-amino group of Lys-80. However, none of the three Gly mutant enzymes was stimulated by exogenously at pH 7.2 (16). The substitutions of Ala for Gly-77 and added ammonia. Presumably, insufficient space is available Gly-78 dramatically decreased thermal stabilities by 4.3 to accommodate an extra ammonia molecule at the active and 5.3 kcal/mol, respectively (Table II). Similar destabil site in the three Gly mutant enzymes because of the presence ization caused by mutations into Ala of conserved glycyl of the side chain of Lys-80. Therefore, one must consider residues was previously observed with adenylate kinase, in other reasons for the low activity only in the oxidative which the G15A and G20A mutant enzymes were more deamination of the G78A mutant enzyme. The larger unstable at high temperatures and more susceptible to

Vol. 116, No. 1, 1994 182 T. Sekimoto et al.

proteolysis than the wild-type enzyme (32). Based on the protein sequence determination, and structural comparison with analysis of the mutant proteins of tryptophan synthase other NAD(P)+-dependent dehydrogenases. Biochemistry 29, ƒ¿-subunit, Ogasahara et al. (33) suggested that suscepti 1009-1015 15. Matsuyama, T., Soda, K., Fukui, T., and Tanizawa, K. (1992) bility to proteolysis is dependent on the conformational Leucine dehydrogenase from Bacillus stearothermophilus: Iden stability of the protein. The present results are fully tification of active-site lysine by modification with pyridoxal consistent with these findings. Thus, the two highly con- phosphate. J. Biochem. 112, 258-265 served glycyl residues (Gly-77 and Gly-78) of leucine 16. Sekimoto, T., Matsuyama, T., Fukui, T., and Tanizawa, K. (1993) Evidence for lysine 80 as general base catalyst of leucine dehydrogenase might also contribute to the conformational dehydrogenase. J. Biol. Chem. 268, 27039-27045 stability of this thermostable enzyme. 17. Sekimoto, T., Fukui, T., and Tanizawa, K. (1994) Involvement of conserved lysine 68 of Bacillus stearothermophilus leucine dehydrogenase in substrate binding. J. Biol. Chem. 269, 7262- REFERENCES 7266 18. Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, 1. Nagata, S., Tanizawa, K., Esaki, N., Sakamoto, Y., Ohshima, T., K.S., Rice, D.W., and Stillman, T.J. (1992) Subunit assembly Tanaka, H., and Soda, K. (1988) Gene cloning and sequence and active site location in the structure of glutamate dehy determination of leucine dehydrogenase from Bacillus stearo drogenase. Proteins: Struct. Funct. Genet. 12, 75-86 thermophilus and structural comparison with other NAD(P)+- 19. Stillman, T.J., Baker, P.J., Britton, K.L., and Rice, D.W. (1993) dependent dehydrogenases. Biochemistry 27, 9056-9062 Conformational flexibility in glutamate dehydrogenase; role of 2. Moon, K., Piszkiewicz, D., and Smith, E.L. 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