Tyrosine 70 increases the coenzyme affinity of aspartate aminotransferase. A site-directed mutagenesis study

The crucial step in enzymatic transamination is the tautomerization of aldimine/ketimine intermediates, formed between the pyridoxyl coenzyme and the amino/keto acid substrate, which is catalyzed primarily by the active site residue Lys-258 (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921; W. L. Finlayson and J. F. Kirsch, in preparation). Tyr-70 is localized in close proximity to Lys-258 and, in addition, forms a hydrogen bond with the coenzyme phosphate. Tyr-70 has been postulated to have an important role in the tautomerization (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525). This hypothesis has now been tested by the construction and analysis of a mutant Escherichia coli aspartate aminotransferase in which Tyr-70 has been changed to Phe (Y70F). Y70F retains at least 15% of the maximal activity of the wild type enzyme (WT) (kcat = 170 +/- 15 s-1 for WT versus greater than or equal to 26 +/- 3 s-1 for Y70F and shows increased Michaelis constants for both substrates (KmAsp = 2.5 +/- 0.4 mM; Km alpha Kg = 0.59 +/- 0.08 mM for WT versus KmAsp = 3.9 +/- 0.3 mM; Km alpha Kg = 2.70 +/- 0.02 mM for Y70F (where alpha Kg is alpha-ketoglutarate) ). The spectrophotometrically determined pK a values of the internal aldimines formed between pyridoxal 5'-phosphate (PLP) and Lys-258 are identical for WT and Y70F. In assays where excess L-aspartate and excess PLP are incubated with either WT or Y70F, the mutant enzyme converts the free PLP to free pyridoxamine 5'-phosphate 80-fold faster than WT (k = (3.75 +/- 0.23) X 10(-2)s-1 for Y70F versus (4.90 +/- 0.02) X 10(-4)s-1 for WT). Y70F also converts free pyridoxamine 5'-phosphate to free PLP faster than WT. Thus, Y70F dissociates coenzyme more readily than does WT. It therefore appears that the role of Tyr-70 is mainly in preventing the dissociation of the coenzyme from the enzyme. Tyr-70 does not function in an essential chemical step.     

A missense mutation (I278T) in the cystathionine beta-synthase gene prevalent in pyridoxine-responsive homocystinuria and associated with mild clinical phenotype.

Cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder characterized by homocystinuria and multisystem clinical disease. Patients responsive to pyridoxine usually have a milder clinical phenotype than do nonresponsive patients, and we studied the molecular pathology of this disorder in an attempt to understand the molecular basis of the clinical variation. We previously reported a T833C transition in exon 8 causing a substitution of threonine for isoleucine at codon 278 (I278T). By PCR amplification and sequencing of exon 8 from genomic DNA we have now detected the I278T mutation in 7 of 11 patients with in vivo pyridoxine responsiveness and in 0 of 27 pyridoxine-nonresponsive patients. Two pyridoxine-responsive patients are homozygous and five are heterozygous for I278T. We have now observed the I278T mutation in 41% (9 of 22) of the independent alleles in pyridoxine-responsive patients of varied ethnic backgrounds. In two of the compound heterozygotes we identified a novel mutation (G139R and E144K) in the other allele. The finding that the two patients who are homozygous for I278T have only ectopia lentis and mild bone demineralization suggests that this mutation is associated with both in vivo pyridoxine responsiveness and mild clinical disease. Compound heterozygous patients who have one copy of this missense mutation are likely to retain some degree of pyridoxine responsiveness.     

Gyrate atrophy of the choroid and retina: characterization of mutant ornithine aminotransferase and mechanism of response to vitamin B6.

The purpose of this study was to characterize the mutant enzyme in nine patients with gyrate atrophy of the choroid and retina associated with ornithine aminotransferase (OAT) deficiency, to elucidate the mechanism of response to pyridoxine in four pyridoxine-responsive patients, and to determine the extent of genetic heterogeneity in both groups of patients. We have measured the apparent Km for pyridoxal phosphate (PLP) in fibroblast mitochondria and the heat stability of OAT at 45 degrees C in the presence and absence of PLP, using a sensitive radiochemical assay. The apparent Km for PLP was higher in pyridoxine-responsive patients than in nonresponsive patients whose apparent Km for PLP was normal. In contrast, the apparent Km for ornithine was normal in the seven patients studied. Surprisingly, the responsive patient with mildest clinical disease had the highest Km for PLP. However, she had the most stable enzyme, which presumably contributed to her milder phenotype. Western blot analyses of mitochondrial proteins, using antibody to human OAT, indicated clearly detectable OAT protein in pyridoxine-responsive patients and in two of five nonresponders, but low or undetectable levels in the other three patients. These data clarify the mechanism of pyridoxine response and indicate heterogeneity within as well as between the pyridoxine-responsive and the nonresponsive patients with gyrate atrophy.     

Studies on the interaction of Escherichia coli agmatinase with manganese ions: structural and kinetic studies of the H126N and H151N variants

The H126N and H151N variants of Escherichia coli agmatinase (EC 3.5.3.11) were produced by site-directed mutagenesis, and their kinetic and structural properties were examined. About 51% and 30% of wild-type activity were expressed by fully manganese activated species of the H126N and H151N variants, respectively. Mutations were not accompanied by changes in the K(m) value for arginine (1.2+/-0.3 mM), K(i) value for putrescine inhibition (3.2+/-0.4 mM), molecular weight (M(r) 67,000+/-2000), tryptophan fluorescence properties (lambda(max) = 342 nm) or CD spectra of the enzyme. However, the interaction with the required manganese ions was significantly altered, as indicated by the effects of dialysis of the enzymes against metal-free buffer. We conclude that replacement of His151 with asparagine results in the loss of a catalytically essential Mn(2+) upon dialysis and concomitant reversible inactivation of the H151N mutant, and that the affinity of a more weakly bound Mn(2+) is decreased in the H126N variant.     

In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus

The reaction velocity of glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) was quantified with a cytophotometer by continuous monitoring of the reaction product as it was formed in liver cryostat sections from normal, young mature female rats at 37 degrees C. Control incubations were performed in media lacking both substrate and coenzyme for G6PDH activity and lacking substrate for PGDH activity. All reaction rates were non-linear but test minus control reactions showed linearity with incubation time up to 5 min using Nitro BT as final electron acceptor. End point measurements after incubation for 5 min at 37 degrees C revealed that the highest specific activity of G6PDH was present in the intermediate area (Vmax = 7.79 +/- 1.76 mumol H2 cm-3 min-1) and of PGDH in the pericentral and intermediate areas (Vmax = 17.19 +/- 1.73 mumol H2 cm-3 min-1). In periportal and pericentral areas, Vmax values for G6PDH activity were 4.48 +/- 1.03 mumol H2 cm-3 min-1) and 3.47 +/- 0.78 mumol H2 cm-3 min-1), respectively. PGDH activity in periportal areas showed a Vmax of 10.84 +/- 0.33 mumol H2 cm3 min-1. Variation of the substrate concentration for G6PDH activity yielded similar KM values of 0.17 +/- 0.07 mM, 0.15 +/- 0.13 mM and 0.22 +/- 0.11 mM in periportal, pericentral and intermediate areas, respectively. KM values of 0.87 +/- 0.12 mM in periportal and of 1.36 +/- 0.10 mM in pericentral and intermediate areas were found for PGDH activity. The significant difference between KM values for PGDH in areas within the acinus support the hypothesis that PGDH is present in the cytoplasmic matrix and in the microsomes. A discrepancy existed between KM and Vmax values determined in cytochemical assays using cryostat sections and values calculated from biochemical assays using diluted homogenates. In cytochemical assays, the natural microenvironment for enzymes is kept for the demonstration of their activity and thus may give more accurate information on enzyme reactions as they take place in vivo.     

Transmural gradients in Na/K pump activity and [Na+]I in canine ventricle

There are well-documented differences in ion channel activity and action potential shape between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) ventricular myocytes. The purpose of this study was to determine if differences exist in Na/K pump activity. The whole cell patch-clamp was used to measure Na/K pump current (I(P)) and inward background Na(+)-current (I(inb)) in cells isolated from canine left ventricle. All currents were normalized to membrane capacitance. I(P) was measured as the current blocked by a saturating concentration of dihydro-ouabain. [Na(+)](i) was measured using SBFI-AM. I(P)(ENDO) (0.34 +/- 0.04 pA/pF, n = 17) was smaller than I(P)(EPI) (0.68 +/- 0.09 pA/pF, n = 38); the ratio was 0.50 with I(P)(MID) being intermediate (0.53 +/- 0.13 pA/pF, n = 19). The dependence of I(P) on [Na(+)](i) or voltage was essentially identical in EPI and ENDO (half-maximal activation at 9-10 mM [Na(+)](i) or approximately -90 mV). Increasing [K(+)](o) from 5.4 to 15 mM caused both I(P)(ENDO) and I(P)(EPI) to increase, but the ratio remained approximately 0.5. I(inb) in EPI and ENDO were nearly identical ( approximately 0.6 pA/pF). Physiological [Na(+)](i) was lower in EPI (7 +/- 2 mM, n = 31) than ENDO (12 +/- 3 mM, n = 29), with MID being intermediate (9 +/- 3 mM, n = 22). When cells were paced at 2 Hz, [Na(+)](i) increased but the differences persisted (ENDO 14 +/- 3 mM, n = 10; EPI 9 +/- 2 mM, n = 10; and MID intermediate, 11 +/- 2 mM, n = 9). Based on these results, the larger I(P) in EPI appears to reflect a higher maximum turnover rate, which implies either a larger number of active pumps or a higher turnover rate per pump protein. The transmural gradient in [Na(+)](i) means physiological I(P) is approximately uniform across the ventricular wall, whereas transporters that utilize the transmembrane electrochemical gradient for Na(+), such as Na/Ca exchange, have a larger driving force in EPI than ENDO.     

Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase

Dimeric mitochondrial aspartate aminotransferase (mAAT) contains a molecule of pyridoxal 5'-phosphate (PLP) tightly attached to each of its two identical active sites. The presence of this natural reporter allows us to study separately local perturbations in the architecture of this critical region of the molecule during unfolding. Upon unfolding of the enzyme with guanidine hydrochloride (GdnHCl), the coenzyme is completely released from the active site. The transition midpoint for the dissociation of PLP is 1.4+/-0.02 M when determined by size-exclusion chromatography (SEC) and 1.6+/-0.02 M when the protein-bound PLP is estimated by electrospray mass spectrometry (ESI-MS). In both cases the transition midpoint is higher than that of inactivation (1.3+/-0.01 M). On the other hand, the midpoint of the unfolding transition obtained by monitoring changes in ellipticity at 356 nm, which reflects the asymmetric environment of the PLP cofactor at the active site, is 1.19+/-0.011 M guanidine. These results indicate that the unfolding of mAAT is a multi-step process which includes an intermediate containing bound PLP but lacking catalytic activity.     

Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, mu = 0.10 m, 25.0 degrees C) revealed wavelength maxima [lambda(max) (nm): 397, 509, approximately equal to 540(sh), 644 (H42A); 404, 516, approximately equal to 538(sh), 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 +/- 0.3 U.mg(-1)) was approximately equal to 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [lambda(max) (nm) = 403, 534, 575(sh), 645] and H42E [lambda(max) (nm) = 404, 530, 573(sh), 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (K(d)) followed by conversion of this intermediate into Compound I (k'(1)). Values for k'(1) and K(d) were, respectively, 4.3 +/- 0.2 s(-1) and 30 +/- 2.0 mM (H42A) and 28 +/- 1.0 s(-1) and 0.09 +/- 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H(2)O(2)] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa = 4.9 +/- 0.1 (rAPX) and pK(a) = 6.7 +/- 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pK(a) for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.     

K dependence of the Na-K pump is abnormal in erythrocytes from patients with cystic fibrosis and obligate heterozygotes

The affinity of the Na-K pump for K was significantly (P less than .001) lower in erythrocytes from patients with cystic fibrosis (Km 4.6 +/- 0.35 mM; n = 26) or from heterozygotes (Km 3.9 +/- 0.57 mM; n = 12) than in controls (Km 2.2 +/- 0.10 mM; n = 20). The affinity of the Na-K pump for K was lower in normal erythrocytes than in normal fibroblasts which may explain the variability in the severity of involvement of different organs in cystic fibrosis. We have now shown in human skin fibroblasts and erythrocytes, that the K affinity of the Na-K pump is lower in patients with cystic fibrosis than in controls. Since the abnormality is also present in erythrocytes from heterozygotes who are clinically normal, it is likely that this abnormality is closely related to the genetic defect in cystic fibrosis.     

Indolylacetic acid and N6-(delta 2-isopentenyl) adenine affect NADH binding to yeast alcohol dehydrogenase and inhibit in vitro the enzymatic oxidation of ethanol

Derivative UV spectroscopic data show that the plant growth substances N6-(delta 2-isopentenyl) adenine (i6Ade) and indolylacetic acid (IAA) can bind to the yeast alcohol dehydrogenase (ADH) and affect coenzyme-enzyme binding. This is confirmed by enzyme kinetics studies. At fixed ethanol concentrations (27.8 and 111.1 mM) and varying NAD+ concentrations (0.033-2 mM), as well as at fixed levels of coenzyme (0.67 and 2 mM), and at varying concentrations of ethanol (1.4-111.1 mM), the rate of ethanol oxidation is significantly inhibited by i6Ade and IAA. The kinetics of the ADH reaction is affected by two inhibition constants (KI and K'I) which correspond to the dissociation constants of complexes EI and ESI, respectively. For i6Ade the KI = 0.52 +/- 0.06 mM and K'I = 0.74 +/- 0.07 mM, and for IAA the KI = 0.88 +/- 0.03 mM and K'I = 0.99 +/- 0.02 mM.     

Entamoeba histolytica: ouabain-insensitive Na(+)-ATPase activity

Our aim was to determine the presence of sodium pumps in Entamoeba histolytica. It is shown through the measurement of ouabain-sensitive ATPase activity and immunoblotting that E. histolytica does not express (Na(+)+K(+))ATPase. On the other hand, we observed a Na(+)-ATPase with the following characteristics: (1) stimulated by Na(+) or K(+), but these effects are not addictive; (2) the apparent affinity is similar for Na(+) and K(+) (K(0.5) = 13.3 +/- 3.7 and 15.4 +/- 3.1mM, respectively), as well as the V(max) (24.9 +/- 1.5 or 27.5 +/- 1.6 nmol Pi mg(-1)min(-1), respectively); (3) insensitive up to 2mM ouabain; and (4) inhibited by furosemide with an IC(50) of 0.12 +/- 0.004 mM. Furthermore, this enzyme forms a Na(+)- or K(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate.     

半胱氨酸脱硫酶突变体H104Q,E156Q,D180G,Q183E和K206A通过改变对磷酸吡哆醛的结合影响酶活性

大肠杆菌半胱氨酸脱硫酶（cysteine desulfurase,IscS）是一类依赖磷酸吡哆醛（pyridoxal phosphate,PLP）的同质二聚体的酶.IscS能催化游离底物L-半胱氨酸脱硫,生成L-丙氨酸和单质硫.在此催化过程中,可形成与酶结合的半胱氨酸过硫化物中间物,并出现了7种具有不同特征性吸收峰的中间反应物.为了研究PLP的结合及中间反应物的形成及累积,对IscS中与PLP结合相关,及IscS半胱氨酸活性口袋中特定氨基酸残基位点（His104,Glu156,Asp180,Gln183和Lys206）进行定点突变,结果发现：1）IscS突变体H104Q、D180G、Q183E、K206A对PLP的结合能力具有不同程度的减弱,酶的活性明显降低甚至消失,PLP与蛋白结合的特异吸收峰消失,或发生明显偏移并出现新的吸收峰,且这些新出现的吸收峰又与蛋白形成的各种中间反应物的吸收峰一致|2）IscS突变体E156Q的活性增高,PLP与蛋白结合的吸收峰明显增加.这些结果都表明,IscS氨基酸残基可通过影响PLP的结合及质子转移引起催化过程中不同中间反应物的形成及累积,同时提高或降低蛋白的活性.     

Reduced ecto-5'-nucleotidase activity and enhanced OKT10 and HLA-DR expression on CD8 (T suppressor/cytotoxic) lymphocytes in the acquired immune deficiency syndrome: evidence of CD8 cell immaturity

Markedly reduced ecto-5'-nucleotidase activity was found in peripheral blood lymphocytes from 27 out of 30 homosexual men with the acquired immune deficiency syndrome (AIDS) in association with Kaposi's sarcoma (AIDS-KS; 2.67 +/- 1.70 U/10(6) cells; n = 13), opportunistic infections (AIDS-OI; 9.29 +/- 7.32; n = 7), or the AIDS-related complex (ARC; 9.82 +/- 6.12; n = 10). These values were significantly different from healthy controls (22.70 +/- 4.58; p less than 0.001). In AIDS-KS patients, both T cells and non-T cells exhibited significantly reduced ecto-5'-NT activity (p less than 0.001). AIDS-KS CD8 cells contained 20% of the mean ecto-5'-NT activity (7.04 +/- 3.53) displayed by control CD8 cells (34.07 +/- 4.86; p less than 0.001). No significant difference in enzyme level was observed between control and AIDS-KS CD4 cells (11.93 +/- 4.98 vs 7.98 +/- 3.28, respectively). In AIDS patients, lymphocyte ecto-5'-NT activity was inversely related (r = -0.518; p less than 0.01) to the absolute number of OKT10+ cells, but no correlation was found with the number of HLA-DR+ cells (r =-0.224). Two-color analysis of lymphocytes from AIDS-KS patients revealed that 75 +/- 12% of circulating CD8 cells expressed the OKT10 antigen, whereas only 10 +/- 6% of control CD8 cells did. HLA-DR antigens, which are not normally found on circulating resting T cells, were expressed in AIDS-KS CD8 cells, although to a lesser extent than OKT10. These data demonstrate that most AIDS CD8 cells differ from control CD8 cells. Although it has been suggested that these cells are activated cytotoxic or suppressor cells, the data presented here support the hypothesis they are immature. Reduced T cell ecto-5'-NT activity and enhanced expression of OKT10 and HLA-DR antigens on circulating CD8 cells, in conjunction with lack of transferrin receptor-(OKT9) and IL 2 receptor-(Tac) bearing lymphocytes, sustain this latter hypothesis. The correlation of the numerical reduction of CD4 cells with the reduced levels of ecto-5'-NT (r = 0.606; p less than 0.01) suggests that the abnormal maturation of CD8 cells seen in AIDS might be a consequence of the CD4 deficiency characteristic of this syndrome.     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 +/- 0.3 versus 1.39 +/- 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 +/- 0.03 versus 0.029 +/- 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 +/- 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the alpha-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.     

Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group

The interaction of Escherichia coli agmatinase (EC 3.5.3.11) with the substrate guanidinium group was investigated by kinetic and site-directed mutagenesis studies. Putrescine and guanidinium ions (Gdn+) were slope-linear, competitive inhibitors with respect to agmatine and their bindings to the enzyme were not mutually exclusive. By site-directed mutagenesis, the E274A variant exhibiting about 1-2% of wild-type activity was obtained. Mutation produced a moderate, but significant, increase in the Km value for agmatine (from 1.1 +/- 0.2 mM to 6.3 +/- 0.3 mM) and the Ki value for competitive inhibition by Gdn+ (from 15.0 +/- 0.1 mM to 44.2 +/- 2.1 mM), but the Ki value for putrescine inhibition (2.8 +/- 0.2 mM) was not altered. The tryptophan fluorescence properties (lambdamax = 342 nm) and circular dichroism spectra were not significantly altered by the Glu274 --> Ala mutation. The dimeric structure of the enzyme was also maintained. We conclude that Glu274 is involved in binding and positioning of the guanidinium moiety of the substrate for efficient catalysis. A kinetic mechanism involving rapid equilibrium random release of products is proposed for E. coli agmatinase.     

Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase

Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5′-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows ∼2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 Å) shows bound ATP, PL and PLP, while D235N (2.3 Å) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz·PLP·ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP.     

Mode of interaction of aminooxy compounds with sheep liver serine hydroxymethyltransferase

The interaction of aminooxy compounds such as aminooxyacetate (AAA), L-canaline, and hydroxylamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) was studied by absorption spectra and stopped-flow spectrophotometry and compared with the unique feature of interaction of O-amino-D-serine (OADS) with the enzyme [Baskaran, N., Prakash, V., Appu Rao, A. G., Radhakrishnan, A. N., Savithri, H. S., & Appaji Rao, N. (1989) Biochemistry (preceding paper in this issue)]. The reaction of AAA (0.5 mM) with the Schiff base of the enzyme resulted in the formation of pyridoxal 5'-phosphate (PLP) and was biphasic with rate constants of 191 and 19 s-1. The formation of the PLP-AAA oxime measured by decrease in absorbance at 388 nm on interaction of AAA with the enzyme had a rate constant of 5.2 M-1 s-1. On the other hand, the reaction of L-canaline with the enzyme was slower as measured by the disruption of enzyme-Schiff base than the reaction of OADS and AAA. In contrast, the formation of PLP as an intermediate could not be detected upon the interaction of hydroxylamine with the enzyme. The reaction of D-cycloserine with the enzyme was much slower (1.6 x 10(2) M-1 s-1) than the aminooxy compounds. These observations indicate that the aminooxy compounds that are structural analogues of serine (OADS, AAA, and canaline) formed PLP as an intermediate prior to the formation of oxime, whereas with hydroxylamine such an intermediate could not be detected.     

Determination of the T-cell subset producing gamma-interferon in tuberculous pleural effusion

The percentage of cells of different T-cell subsets and their functions in tuberculous pleural effusion were investigated. The percentage of OKT4-positive cells was 65 +/- 2% (mean +/- SEM, n = 8) and that of OKT8-positive cells was 19 +/- 3% (n = 8). Pleural T lymphocytes of patients with tuberculous pleurisy responded well to stimulation with purified protein derivative of tuberculin, and deoxyribonucleic acid synthesis was observed along with gamma interferon (IFN-gamma) production. When pleural T lymphocytes of patients with tuberculous pleurisy were treated with OKT4 monoclonal antibody and complement, a significant decrease in IFN-gamma production was observed in all eight patients (P less than 0.005), whereas no definite decrease in IFN-gamma production was found after treatment with OKT8 monoclonal antibody and complement. These results suggest that at least the OKT4+/OKT8- T-cell subset is responsible for the antigen-specific IFN-gamma production in pleural T lymphocytes of patients with tuberculous pleurisy.     

Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies. Alteration of substrate specificity by replacement of Asn149 with Asp

To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The K(m) for arginine (1.4-1.6 mM) was not altered and the wild-type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (K(m) = 2.5 +/- 0.2 mM) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn(2+) and contained < 0.1 Mn(2+).subunit(-1), whereas wild-type and His145Asn enzymes were half active and contained 1.1 +/- 0.1 Mn(2+).subunit(-1) and 1.3 +/- 0.1 Mn(2+).subunit(-1), respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with K(d) of 1.8 +/- 0.2 x 10(-8) M for the wild-type and His145Asn enzymes and 16.2 +/- 0.5 x 10(-8) m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (lambda(max) = 338-339 nm) and sensitivity to thermal inactivation were not altered. The Asn149-->Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn(2+) (A) is the more tightly bound metal ion in arginase II.