Binding of pyridoxal 5'-phosphate to the heme protein human cystathionine beta-synthase

Cystathionine beta-synthase (CBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme, catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS was recently shown to be a heme protein. While the role of heme in CBS is unknown, catalysis by CBS can be explained solely by participation of PLP in the reaction mechanism. In this study, treatment of CBS with sodium borohydride selectively reduced the Schiff base but did not affect the heme. Purification and sequencing of the PLP-cross-linked peptide from a trypsin digest of the reduced enzyme revealed the evolutionarily conserved Lys119 to be the residue forming the Schiff base. Serine and hydroxylamine form an alpha-aminoacrylate and an oxime with PLP in CBS, respectively. The sulfhydryl-containing substrate, homocysteine, disturbs the heme environment but does not interact with PLP. In contrast to other PLP-dependent enzymes, CBS emits no PLP-related fluorescence when excited at 296 or 330 nm. PLP but not heme dissociates from the enzyme in the presence of hydroxylamine. The dissociation of PLP is a multistage process involving a short approximately 500 s lag phase, followed by a rapid inactivation and a slower PLP-oxime formation. PLP-free CBS exhibits a decrease of secondary structure as well as loss of CBS activity that can be only partially restored by PLP. This study constitutes the first comprehensive investigation of PLP interaction with a heme protein.     

Myelin Proteolipid Protein Contains Thioester-Linked Fatty Acids

Myelin proteolipid protein (PLP) is known to contain long-chain, covalently bound fatty acids. Previous studies, including our own, have suggested the occurrence of an oxyester type of linkage between fatty acids and PLP. However, we found that protein-SH groups are required in the acylation reaction, suggesting the possible presence of thioesters. In the present study, we have examined the nature of the acyl-PLP linkages by determining whether free thiol groups are generated on removal of fatty acids. Incubation of reduced and carboxyamidomethylated proteolipid apoprotein (RCM-APL) with 0.2 M hydroxylamine and [14C]iodoacetamide at pH 7.5 and 37 degrees C resulted in the release of fatty acids and the concomitant labeling of newly formed thiol groups. Incubation with Tris or methylamine at pH 7.5 failed to remove fatty acids and generate free -SH groups. The possibility that on treatment buried thiol groups became exposed was essentially excluded because (1) similar results were obtained in 2-chloroethanol, a solvent in which acylated and deacylated PLP have the same conformation, and (2) small PLP peptides were labeled only in the presence of hydroxylamine. On incubation with [14C]methylamine at pH 9.0, RCM-APL was not labeled, thus excluding the occurrence of intramolecular thiol esters. On the other hand, fatty acids were released as radioactive N-methyl fatty acylamide, indicating the presence of intermolecular thioesters between fatty acids and protein. These results demonstrate that a large proportion of fatty acids covalently bound to PLP are liked to -SH groups.     

Discovery and characterization of d-phenylserine deaminase from Arthrobacter sp. TKS1

We discovered a D-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from D-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55°C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50°C. The enzyme acted on the D-forms of β-hydroxy-α-amino acids, such as D-threo-phenylserine (K(m), 19 mM), D-serine (K(m), 5.8 mM), and D-threonine (K(m), 102 mM). As L-threonine, D-allo-threonine, L-allo-threonine, and DL-erythro-phenylserine were inert, the enzyme could distinguish D-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO(4), CuSO(4), BaCl(2), and CoCl(2) and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and DL-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.     

The effect of pyridoxal phosphate on the tryptophanases of five species of Enterobacteriaceae]

Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris et P. morganii tryptophanases (TPases) were studied for the spectral forms of the enzyme. The pH effect on the absorption spectrum and on the enzyme specific activity revealed that the coli group TPases are identical with but differ from Proteus TPases which differ themselves. The coli group TPases attach 4 mol of pyridoxal phosphate (PLP)/mol of enzyme, independently of the pH in the presence of K(plus) ions, and 9 mol of PLP/mol of enzyme must be reduced to achieve complete inactivation. The Proteus TPases attach 4 mol of PLP/mol of enzyme at PH 6.8, and 3 mol of PLP/mol of enzyme at pH 7.8 in K(plus) buffer. In P. morganii, 7 mol of PLP/mol of enzyme must be reduced to inactivate the enzyme, whereas P. vulgaris TPase cannot be completely inactivated by this method. These five TPases attach only 3 mol of PLP/mol of enzyme in a Na(plus) buffer, independently of the pH.     

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.     

Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites

Cystathionine beta-synthase is an unusual enzyme that requires the cofactors heme and pyridoxal phosphate (PLP) to catalyze the condensation of homocysteine and serine to generate cystathionine. This transsulfuration reaction represents one of two major cellular routes for detoxification of homocysteine, which is a risk factor for atherosclerosis. While the beta-replacement reaction catalyzed by this enzyme suggests a role for the pyridoxal phosphate, the role of the heme is uncertain. In this study we have examined the effect of changing one of the ligands to the heme on the activity of the enzyme. Binding of carbon monooxide results in the displacement of a thiolate ligand to the ferrous heme, and is accompanied by complete loss of cystathionine beta-synthase activity. Furthermore, inhibition by CO is competitive with respect to homocysteine, providing the first indication that the homocysteine binding site is in the proximity of heme. Binding of both CO and cyanide to ferrous cystathionine beta-synthase occurs in two distinct isotherms and indicates that the hemes are nonequivalent. We have employed fluorescence spectroscopy to characterize the bound PLP and its interaction with serine. PLP bound to cystathionine beta-synthase is weakly fluorescent and exists as a mixture of the protonated and unprotonated tautomers. Reaction with hydroxylamine releases the oxime and greatly enhances the associated fluorescence. Binding of serine is accompanied by a shift to the unprotonated tautomer of the external aldimine as well as the appearance of a new fluorescent species at approximately 400 nm that could be due to the aminoacrylate or to a gemdiamine intermediate. These data provide the first characterization of the PLP bound to cystathionine beta-synthase. Treatment of cystathionine beta-synthase with hydroxylamine releases two PLPs after 1 day and results in complete loss of activity. Incubation for an additional 3-4 days results in the release of two more PLPs. These data lead us to revise the PLP stoichiometry to 4 per tetramer, and to the conclusion that the heme and PLP sites in cystathionine beta-synthase are nonequivalent.     

The ionization states of the 5'-phosphate group in the various coenzyme forms bound to mitochondrial aspartate aminotransferase

We have carried out a Fourier transform infrared spectroscopic study of mitochondrial aspartate aminotransferase in the spectral region where phosphate monoesters give rise to absorption. Infrared spectra in the above-mentioned region are dominated by protein absorption. Yet, below 1020 cm-1 protein interferences are minor, permitting the detection of the band arising from the symmetric stretching of dianionic phosphate monoesters [T. Shimanouchi, M. Tsuboi, and Y. Kyogoku (1964) Adv. Chem. Phys. 8, 435-498]. The integrated intensity of this band in several enzyme forms (pyridoxal phosphate, pyridoxamine phosphate, and sodium borohydride-reduced, pyridoxyl phosphate form) does not change with pH in the range 5-9. This behavior contrasts that of free pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) in solution, where the dependence of the same infrared band intensity with pH can be correlated to the known pK values for the 5'-phosphate ester in solution. The integrated intensity value of this infrared band for the PLP enzyme form before and after reduction with sodium borohydride is close to that given by free PLP at pH 8-9. These results are taken as evidence that in the active site of mitochondrial aspartate aminotransferase the 5'-phosphate group of PLP remains mostly dianionic even at a pH near 5. Thus, it is suggested that the chemical shift changes associated with pH titrations of various PLP forms reported in a previous 31P NMR study of this enzyme [M. E. Mattingly, J. R. Mattingly, and M. Martinez-Carrion (1982) J. Biol. Chem. 257, 8872] are due to the fact that the phosphorus chemical shift senses the O-P-O bond distortions induced by the ionization of a nearby residue. Since no chemical shift changes were observed in pH titrations of the PMP forms (lacking an ionizable internal aldimine) of this isozyme, the Schiff base between PLP and Lys-258 at the active site is the most likely candidate for the ionizing group influencing the phosphorus chemical shift in this enzyme.     

Some properties of serine: glyoxylate aminotransferase from rye seedlings (Secale cereale L.).

Serine: glyoxylate aminotransferase (EC 2.6.1.45) from rye seedlings catalysed transamination between L-serine and glyoxylate according to the Ping Pong Bi Bi mechanism with double substrate inhibition. As judged from the Km values, L-serine, L-alanine, and L-asparagine served as substrates for the enzyme with glyoxylate, whereas L-alanine and L-asparagine underwent transamination with hydroxypyruvate as acceptor. Pyridoxal phosphate (PLP) seems to be rather loosely bound to the enzyme protein. Aminooxyacetate and D-serine were found to be pure competitive inhibitors of the enzyme, with Ki values of 0.12 microM and 1.6 mM, respectively. Among the PLP inhibitors isonicotinic acid hydrazide and hydroxylamine were far less effective than aminooxyacetate (20% and 70% inhibition at 0.1 mM concentration, respectively). Inhibition by the SH group inhibitors at 1 mM concentration did not exceed 50%. L-Serine distinctly diminished the inhibitory effect of this type inhibitors. Preincubation of the enzyme with glyoxylate distinctly diminished transamination. Glyoxylate limited the inhibitory action of formaldehyde probably by competing for the reactive groups present in the active centre.     

Chemical deacylation reduces the adhesive properties of proteolipid protein and leads to decompaction of the myelin sheath

Myelin proteolipid protein (PLP) contains thioester-bound, long-chain fatty acids which are known to influence the structure of the molecule. To gain further insights into the role of this post-translational modification, we studied the effect that chemical deacylation of PLP had on the morphology of myelin and on the protein's ability to mediate the clustering of lipid vesicles. Incubation of rat optic nerves in isoosmotic solutions containing 100 mM hydroxylamine (HA) pH 7.4 led to deacylation of PLP and decompaction of myelin lamellae at the level of the intraperiod line. Incubation of nerves with milder nucleophilic agents (Tris and methylamine) or diluted HA, conditions that do not remove protein-bound fatty acids, caused no alterations in myelin structure. Other possible effects of HA which could have affected myelin compaction indirectly were ruled out. Incubation of optic nerves with 50 mM dithioerythritol (DTE) also led to the splitting of the myelin intraperiod line and this change again coincided with the removal of fatty acids. In addition, the apparently compacted CNS myelin in the PLP-less myelin-deficient rat, like that in tissue containing deacylated PLP, was readily decompacted upon incubation in isoosmotic buffers, suggesting that the function of PLP as a stabilizer of the interlamellar attachment is, at least in part, mediated by fatty acylation. Furthermore, in contrast to the native protein, PLP deacylated with either HA or DTE failed to induce the clustering of phosphatidylcholine/cholesterol vesicles in vitro. This phenomenon is not due to side-effects of the deacylation procedure since, upon partial repalmitoylation, the protein recovered most of its original vesicle-clustering activity. Collectively, these findings suggest that palmitoylation, by influencing the adhesive properties of PLP, is important for stabilizing the multilamellar structure of myelin.     

Polypyrroles formed from porphobilinogen and amines by uroporphyrinogen synthetase of Rhodopseudomonas spheroides

1. Uroporphyrinogen I synthetase of Rhodopseudomonas spheroides was purified more than 200-fold from the soluble protein of broken bacterial cells. The enzyme had molecular weight 36000, an isoelectric point of 4.46 and migrated as a single active protein band on disc-gel electrophoresis at pH7.5 and 8.9. 2. The enzyme consumed porphobilinogen and formed uroporphyrinogen at pH8.2 without the accumulation of intermediates. In the presence of hydroxylamine, ammonia or methoxyamine the production of porphyrinogen was inhibited and the enzyme formed open-chain polypyrroles instead. 3. These polypyrroles behaved like uroporphyrinogen on Sephadex G-25; they were colourless and had unsubstituted alpha-pyrrolic positions. The inhibitory amines were incorporated into the molecules. 4. The polypyrroles formed porphyrins non-enzymically and the cyclization reaction was accompanied by the release of the inhibitory amine. Exchange of the amino function of the original porphobilinogen in the polypyrrole was complete with hydroxylamine and almost complete with methoxyamine, both ammonia and methoxyamine being present in the polypyrrolic material. 5. The behaviour, properties and composition of the radioactive hydroxylamine derivative were consistent with a tetrapyrrolic structure, probably a pyrrylmethane, that was not cyclized, rather than with di-, tri- or penta-pyrrolic structures. No monopyrrolic or dipyrrolic Ehrlich-positive material was released on cyclization. The ammonia and methoxyamine derivatives had properties similar to the hydroxylamine derivative. 6. Another modified pyrrole was detected only in experiments with hydroxylamine. It differed from both porphobilinogen and known dipyrroles and appeared to be a monopyrrole. 7. The participation of positively charged reaction centres in the enzymic mechanism, particularly in the cyclization step, is discussed.     

pH dependence of the absorbance and 31P NMR spectra of O-acetylserine sulfhydrylase in the absence and presence of O-acetyl-L-serine.

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the final step in the biosynthesis of L-cysteine in Salmonella, viz., the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. UV-visible spectra of OASS exhibit absorbance maxima at 280 and 412 nm with pH-independent extinction coefficients over the range 5.5-10.8. Addition of OAS to enzyme results in a shift in the absorbance maximum from 412 to 470 nm, indicating the formation of an alpha-aminoacrylate Schiff base intermediate [Cook, P. F., & Wedding, R. T. (1976) J. Biol. Chem. 251, 2023]. The spectrum of the intermediate is also pH independent from 5.5 to 9.2. The observed changes in absorbance at 470 nm at different concentrations of OAS were used to calculate a Kd of 3 microM for OAS at pH 6.9. As the pH decreases, the Kd increases an order of magnitude per pH unit. The 31P NMR signal of the bound PLP has a pH-independent chemical shift of 5.2 ppm in the presence and absence of OAS. These results indicate that the phosphate group is present as the dianion possibly salt-bridged to positively charged groups of the protein. In agreement with this, the resonance at 5.2 ppm has a line width of 20.5 Hz, suggesting that the cofactor is tightly bound to the protein. The sulfhydrylase was also shown to catalyze an OAS deacetylase activity in which OAS is degraded to pyruvate, ammonia, and acetate. The activity was detected by a time-dependent disappearance of the 470-nm absorbance reflecting the alpha-aminoacrylate intermediate. The rate of disappearance of the intermediate was measured at pH values from 7 to 9.5 using equal concentrations of OAS and OASS. The rate constant for disappearance of the intermediate decreases below a pK of 8.1 +/- 0.1, reflecting the deprotonation of the active-site lysine that originally formed the Schiff base with PLP in free enzyme. A possible mechanism for the deacetylase activity is presented where the lysine displaces alpha-aminoacrylate which decomposes to pyruvate and ammonia.     

Inhibition of acetyl-CoA carboxylase isoforms by pyridoxal phosphate

Mammalian isoforms of acetyl-CoA carboxylase (ACC-1 and ACC-2) play important roles in synthesis, elongation, and oxidation of long-chain fatty acids, and the possible significance of ACC in the development of obesity has led to interest in the development of inhibitors. Here, we demonstrate that pyridoxal phosphate (PLP) is a linear and reversible inhibitor of ACC-1 and ACC-2. ACC from rat liver and white adipose tissue (largely ACC-1) exhibited an IC50 of approximately 200 microm, whereas ACC-2 from heart or skeletal muscle exhibited an IC50 exceeding 500 microm. ACC from rat liver was equally sensitive to PLP following extensive purification by avidin affinity chromatography. When added before citrate, PLP inhibited ACC with a Ki of approximately 100 microm, reducing maximal activity >90% and increasing the Ka for citrate approximately 5-fold but having little effect on substrate Km values. Pre-treatment with citrate increased the apparent Ki for ACC inhibition by PLP by approximately 4-fold. Inhibition of ACC was reversed by removal of PLP, either by washing or by reaction with hydroxylamine or amino-oxyacetate. ACC was irreversibly inhibited and radiolabeled, to a stoichiometry of approximately 0.4 mol[H]/mol subunit, in the presence of PLP plus [3H]borohydride. Studies with structurally related compounds demonstrated that the reactive aldehyde and negatively charged substituents of PLP contribute importantly to ACC inhibition. The studies reported here suggest a rationale to develop ACC inhibitors that are not structurally related to the substrates or products of the reaction and an approach to probe the citrate-binding site of the enzyme.     

Characterization of proteolipid protein fatty acylesterase from rat brain myelin.

A protein fatty acylesterase activity that catalyzes the removal of fatty acid from exogenous proteolipid protein (PLP) has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the deacylation of PLP was obtained in 0.5% Triton X-100, 1 mM dithiothreitol at pH 7.0 and at 37 degrees C. Other detergents (octyl beta-D-glucoside, Nonidet P-40, and Tween 20) have little or no effect, whereas deacylation was completely abolished by 0.1% sodium dodecyl sulfate or boiling the membrane fraction for 5 min prior to incubation. Under optimal conditions, the rate of deacylation was linear up to 20 min, and the apparent Km for bovine [3H]palmitoyl-PLP was 18 microM. The myelin-associated PLP fatty acylesterase has no apparent requirements for divalent cations (Ca2+, Mg2+, Mn2+), and chelators such as EDTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, and 1,10-phenantroline have little or no effect on enzyme activity. Sulfhydryl and histidine residues are needed for full enzyme activity, whereas the "active serine"-directed inhibitor phenylmethylsulfonyl fluoride has no effect. The myelin-associated protein fatty acylesterase was present throughout brain development and in all myelin subfractions, in agreement with the dynamic metabolism of PLP-bound fatty acids. Enzyme activity was also present in sciatic nerve, brain cortex, and heart whereas liver was devoid of activity. Several esterases, including phospholipase A2, glyoxalase II, and acetylcholinesterase, did not remove fatty acid from PLP. Myelin basic protein, palmitoyl-CoA hydrolase, and myelin-associated nonspecific esterase were also ruled out as the PLP fatty acylesterase. Thus, all data seem to indicate that this enzyme is different from esterases of the lipid metabolism. Finally, stimulation of protein phosphorylation with Ca2+, but not with cyclic-AMP, inhibited PLP deacylation, suggesting that the myelin-associated protein fatty acylesterase activity is regulated by endogenous Ca(2+)-dependent protein kinases.     

The nature of phosphate residue binding in the phosphorylated form of succinyl-CoA-synthetase from pigeon breast muscle

The hydrolytic stability of phosphorylated pigeon breast muscle succinyl-CoA synthetase within a wide pH range was studied. It was found that within complex I the phosphate-protein bond is hydrolyzed at alkaline values of pH (11.0 and 13.0); at acidic pH values this bond is hydrolyzed by 50%. Within complex II the phosphate-protein bond is hydrolyzed at acidic pH values and is stable at alkaline pH values. The reaction of the phosphorylated enzyme with hydroxylamine and diisopropylfluorophosphate results in protein dephosphorylation by 50%. Ion-exchange chromatography of the radioactive phosphorylated enzyme II alkaline hydrolyzate (3 n NaOH, 3 hours, 100 degrees C) revealed that the radioactivity was distributed between 1-N-, 3-N-phosphohistidine and 1.3-N-diphosphohistidine fractions. The experimental results suggest that in the phosphorylated enzyme I phosphate is bound to the protein to form an acyl phosphate and phosphoester bonds, while in the phosphorylated enzyme II phosphate binding to the protein occurs with the formation of phosphoamide bonds.     

Resonance Raman spectra of the pyridoxal coenzyme in aspartate aminotransferase. Evidence for pyridine protonation and a novel photochemical H/D exchange at the imine carbon atom

Resonance Raman (RR) spectra are reported for aspartate aminotransferase from pig heart cytosol, and for inhibitor complexes. They are interpreted with reference to the previously analyzed spectra of pyridoxal phosphate (PLP) Schiff base adducts. This comparison shows that, as expected, the pyridine N atom is protonated in the native enzyme at pH 5, and in the glutarate complexes at pH 8.5, and that it is also protonated in the alpha-methylaspartate complex; the stabilization of the pyridine proton at high pH must be due to the interaction with aspartate 222 seen in the x-ray crystal structure. RR spectra of the erythro-beta-hydroxy-DL-aspartate complex, representing the p-quinoid enzyme intermediate, as well as of AlIII complexes of PLP Schiff bases with phenylalanine and tyrosine ethyl ester have been obtained via the coherent anti-Stokes Raman scattering technique, and partially assigned. A novel H/D exchange at the coenzyme C4' atom has been observed for the native enzyme in D2O, and has been determined, by a combination of NMR and RR measurements, to be due to the Raman laser irradiation. This photoprocess, which is not observed for PLP Schiff bases in aqueous solution, is attributed to a photoexcited p-quinoid intermediate, similar to that implicated in the enzyme mechanism. It is suggested that this intermediate is stabilized by protein interactions which localize charge on the phenolate O atom, plausibly a hydrogen bond from the nearby tyrosine 225. H/D exchange would then follow via the aldimine-ketimine interconversion known to take place in the enzyme reaction.     

Structural basis of the tight binding of pyridoxal 5'-phosphate to a low molecular weight protein tyrosine phosphatase

Pyridoxal 5'-phosphate (PLP) binds tightly to bovine low Mr protein tyrosine phosphatase (BPTP), but it is a very poor substrate for the enzyme. The structural basis of this tight binding of PLP is examined here by a variety of methods. Binding constants of a number of PLP analogues were measured with wild-type BPTP, and PLP binding constants of some site-specific mutants of BPTP were determined at pH 5.0 through the use of several independent methods. The tight binding of PLP (Ki = 7.6 microM) causes a downfield shift of the His-72 Cepsilon1H resonance in the 1H NMR spectrum of the protein, consistent with a structural alteration in the phosphate binding loop transmitted through a complex hydrogen bond network that exists between His-72 and Asn-15, which is a residue in the phosphate binding loop. 1H NMR spectroscopy with an MLEV-17 spectral editing scheme was used to monitor the aldehyde resonance of PLP during titration of a catalytically inactive C12A mutant of BPTP. The aldehydic proton resonance of PLP shifted from 10.43 to 10.26 ppm upon complex formation with the C12A mutant. This resonance occurs far from the region where a hemithioacetal hydrogen would be expected to appear, consistent with the conclusion that the Cys-17 side chain of BPTP does not add to the aldehyde group of PLP. UV-visible spectrophotometric titration also supported this conclusion. The binding constant of PLP to a C17A mutant was similar to that exhibited with wild-type protein. These results show that Cys-17 makes virtually no contribution to the tight binding of PLP by BPTP, in contrast to a published report that it is "essential" for binding PLP. On the other hand, Asp-129 of BPTP was found to be very important for binding PLP. It is concluded that Asp-129 binds to the pyridinium nitrogen of PLP and that this renders Asp-129 effectively unavailable to serve its essential catalytic role as a general acid. The interactions described here should be useful in the design of specific inhibitors of this and related phosphotyrosyl protein phosphatases.     

P NMR studies of O-acetylserine sulfhydrylase-B from Salmonella typhimurium

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine and bisulfide to l-cysteine and acetate in bacteria and higher plants. Enteric bacteria have two isozymes of OASS, A and B, produced under aerobic and anaerobic growth conditions, respectively, with different substrate specificities. The ³¹P chemical shift of the internal and external Schiff bases of PLP in OASS-B are further downfield compared to OASS-A, suggesting a tighter binding of the cofactor in the B-isozyme. The chemical shift of the internal Schiff base (ISB) of OASS-B is 6.2 ppm, the highest value reported for the ISB of a PLP-dependent enzyme. Considering the similarity in the binding sites of the PLP cofactor for both isozymes, torsional strain of the C5-C5′ bond (O4′-C5′-C5-C4) of the Schiff base is proposed to contribute to the further downfield shift. The chemical shift of the lanthionine external Schiff base (ESB) of OASS-B is 6.0 ppm, upfield from that of unliganded OASS-B, while that of serine ESB is 6.3 ppm. Changes in chemical shift suggest the torsional strain of PLP changes as the reaction proceeds.The apoenzyme of OASS-B was prepared using hydroxylamine as the resolving reagent. Apoenzyme was reconstituted to holoenzyme by addition of PLP. Reconstitution is pseudo-first order and exhibits a final maximum recovery of 81.4%. The apoenzyme shows no visible absorbance, while the reconstituted enzyme has a UV-visible spectrum that is nearly identical to that of the holoenzyme. Steady-state fluorescence spectra gave tryptophan emission of the apoenzyme that is 3.3-fold higher than the emission of either the native or reconstituted enzyme, suggesting that PLP is a potent quencher of tryptophan emission.     

Cysteine-108 is an acylation site in myelin proteolipid protein.

Myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids. A large proportion of these acyl moieties are bound in thioester linkages, as demonstrated by alkylation of newly formed SH groups upon deacylation. To identify the Cys residue(s) involved in the thioester linkage(s), reduced and carboxyamidomethylated proteolipid protein was labeled with [14C]iodoacetamide upon deacylation with neutral hydroxylamine. The labeled protein was digested with trypsin or pepsin, and peptides analyzed by RP-HPLC. Identification of the isolated radioactive peptides by amino acid analysis, peptide sequencing and/or fast-atom bombardment-mass spectrometry revealed that Cys108 in the bovine PLP sequence is an acylated site. The sequence surrounding the palmitoylation site in the myelin PLP is strikingly similar to that found in rhodopsin. Furthermore, as in rhodopsin and other members of the G protein-coupled receptor family, this Cys residue is located within a hydrophilic, basic, and possibly cytoplasmic, domain.     

Unusual structural,functional, and stability properties of serine hydroxymethyltransferase from Mycobacterium tuberculosis

From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5'-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with d-alanine resulting in formation of apoenzyme but l-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. Urea- and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations.     

Characterization of a phosphoenzyme intermediate in the reaction of phosphoglycolate phosphatase

When 32P-glycolate and phosphoglycolate phosphatase from spinach are mixed, 32P is incorporated into acid precipitated protein. Properties that relate the phosphorylation of the enzyme to the phosphatase are: the Km value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the 32P in the phosphoenzyme is diluted by unlabeled P-glycolate or the specific alternative substrate, ethyl-P; the activator Cl- enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of 32P-enzyme; and 32P is lost from the enzyme when 32P-glycolate is consumed. The phosphorylated protein has a molecular weight of 34,000, which is half that of the native protein and is similar in size to the labeled band that is seen on sodium dodecyl sulfate-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acylphosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by 32P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms (Rose, Z. B., Grove, D. S., and Seal, S. N. (1986) J. Biol. Chem. 261, 10996-11002).