Biomedical subjects

F C Hartman

Publications and source records attributed to F C Hartman.

At least 55 records · Page 3Linked to original sources

PubMed1989

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site.

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.

Affinity Labels↗

PubMed1989

Examination of subunit interactions at the active site of ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum by hybridization of site-directed mutants.

The two active sites of homodimeric ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum are constituted by interacting domains of adjacent subunits, in which residues from each are required for catalytic activity. Active-site residues include Lys-166 of one domain and Glu-48 of the interacting domain from the adjacent subunit. Whereas all substitutions for Lys-166, introduced by site-directed mutagenesis, abolished catalytic activity, only a negatively charged residue (e.g., aspartic acid) resulted in the disruption of the subunit interactions (Lee et al., 1987). This disruption could result from improper folding of the individual polypeptide chains or to more localized effects (e.g., charge-charge repulsion due to proximal negative charges of Asp-166 and Glu-48 of adjacent domains or conformational changes restricted to a single domain). To address these questions, we have examined the ability of the Asp-166 mutant subunit to associate with a mutant subunit in which the negatively charged Glu-48 has been replaced by the neutral glutaminyl residue. Coexpression in Escherichia coli of the genes for both mutant subunits results in formation of a catalytically active hybrid, despite the absence of activity when either gene is expressed individually. Isolation and characterization of the hybrid show that it is composed of one Asp-166 subunit and one Gln-48 subunit, presumably with only one functional active site per dimeric molecule. This association of dissimilar subunits shows that introduction of a negative charge at position 166 does not lead to overall distortion of subunit conformation. In contrast to the wild-type enzyme, the hybrid dissociates spontaneously at low protein concentration but is stabilized by elevated ionic strengths or by glycerol.

Binding Sites↗

PubMed1988

Catalytic nonessentiality of an active-site cysteinyl residue of phosphoribulokinase.

The activity of the Calvin cycle enzyme phosphoribulokinase is coupled to photosynthetic electron transport by reversible oxidation/reduction mediated by thioredoxin-f. Previous studies have shown that one of the regulatory sulfhydryl groups, that of Cys-16, is positioned at the nucleotide-binding domain of the active site. To determine if oxidative deactivation of the kinase reflects catalytic essentiality of Cys-16, the methylation of spinach phosphoribulokinase by methyl-4-nitrobenzenesulfonate has been examined. Methylation of the kinase results in a 50% loss of the initial activity relative to controls. The suppression of kcat is accompanied by a 6-fold increase in the Km for ATP, without change in the Km for ribulose 5-phosphate. The insensitivity of the modified enzyme, in contrast to the native, to iodoacetate and 5,5'-dithiobis(2-nitrobenzoate) indicates that Cys-16 is a site of methylation. This supposition is verified independently by peptide mapping and Edman degradation subsequent to S-carboxymethylation with [14C]iodoacetate of the methylated kinase. Retention of significant enzymatic activity after complete modification of Cys-16 with the small, uncharged methyl moiety demonstrates that this active-site residue is not essential for catalysis.

Benzenesulfonates↗

PubMed1988

Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis.

The epsilon-amino group of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase was postulated as the essential base which initiates catalysis by abstracting the proton at C-3 of ribulose 1,5-bisphosphate (Hartman, F. C., Soper, T. S., Niyogi, S. K., Mural, R. J., Foote, R. S., Mitra, S., Lee, E. H., Machanoff, R., and Larimer, F. W. (1987) J. Biol. Chem. 262, 3496-3501). To scrutinize this possibility, the site-directed Gly-166 mutant, totally devoid of ribulosebisphosphate carboxylase activity, was examined for its ability to catalyze each of three partial reactions. When carbamylated at Lys-191 (i.e. activated with CO2 and Mg2+), wild-type enzyme catalyzed the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon reaction intermediate of the carboxylase reaction (Pierce, J., Andrews, T. J., and Lorimer, G. H. (1986a) J. Biol. Chem. 261, 10248-10256). Likewise, when carbamylated at Lys-191, the Gly-166 mutant also catalyzed the hydrolysis of this reaction intermediate. The carbamylated wild type catalyzed the enolization of ribulose 1,5-bisphosphate as indicated by the transfer of 3H radioactivity from [3-3H]ribulose, 1,5-bisphosphate to the medium. However, even when carbamylated at Lys-191, the mutant protein did not catalyze the enolization of ribulose 1,5-bisphosphate. Additionally, unlike the decarbamylated wild-type enzyme, which catalyzed the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate in the absence of Mg2+, the mutant protein was inactive in this partial reaction. These properties exclude the epsilon-amino group of Lys-166 as an obligatory participant in the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. In contrast, these properties are consistent with the epsilon-amino group of Lys-166 functioning as an acid-base catalyst in the enolization of ribulose 1,5-bisphosphate (when the enzyme is carbamylated) and in the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (when the enzyme is decarbamylated). Alternatively, Lys-166 may stabilize the transition states of these two partial reactions.

Binding Sites↗

PubMed1988

Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification.

Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.

Amino Acid Sequence↗

PubMed1988

Restoration of activity to catalytically deficient mutants of ribulosebisphosphate carboxylase/oxygenase by aminoethylation.

Substitutions for active-site lysyl residues at positions 166 and 329 in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been shown to abolish catalytic activity. Treatment of the Cys-166 and Cys-329 mutant proteins with 2-bromoethylamine partially restores enzyme activity, presumably as a consequence of selective aminoethylation of the thiol group unique to each protein. Amino acid analyses, slow inactivation of the wild-type carboxylase by bromoethylamine, and the failure of bromoethylamine to restore activity to the corresponding glycyl mutant proteins support this interpretation. The observed facile, selective aminoethylations may reflect an active site microenvironment not dissimilar to that of the native enzyme. Catalytic constants of these novel carboxylases, which contain a sulfur atom in place of a specific lysyl gamma-methylene group, are significantly lower than that of the wild-type enzyme. Furthermore, the aminoethylated mutant proteins form isolable complexes with a transition state analogue, but with compromised stabilities. These detrimental effects by such a modest structural change underscore the stringent requirement for lysyl side chains at positions 166 and 329. In contrast, the aminoethylated mutant proteins exhibit carboxylase/oxygenase activity ratios and Km values that are unperturbed relative to those for the native enzyme.

Binding Sites↗

PubMed1988

Characterization of the regulatory thioredoxin site of phosphoribulokinase.

Phosphoribulokinase is light-regulated via thioredoxin by reversible oxidation/reduction of sulfhydryl/disulfide groups. To identify the cysteinyl residues that are involved in regulation, the S-carboxymethyl labeling patterns of the fully reduced (active) and oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced, [14C]carboxymethylated enzyme contained four labeled peptides, all of which were purified and sequenced by Edman degradation. If the enzyme was oxidized by 5,5'-dithiobis-(2-nitrobenzoic acid) prior to carboxymethylation and tryptic digestion, only two labeled peptides were observed, thereby revealing the identity of the regulatory cysteines as Cys-16 and Cys-55. The former was previously implicated as part of the nucleotide-binding domain of the active site (Porter, M.A., and Hartman, F.C. (1986) Biochemistry 25, 7314-7318), a conclusion reinforced by the present observation that the sequence around the Cys-16 is similar to a consensus sequence of ATP-binding sites from a number of proteins of diverse phylogenetic origin (Higgins, C.F., Hiles, I.D., Salmond, G.P.C., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W., and Hermondson, M. (1986) Nature 323, 448-450). The regulatory disulfide of phosphoribulokinase was found to be intrasubunit based on the stoichiometry of the oxidation and the failure to resolve oxidized and reduced enzyme by gel filtration under dissociation conditions.

Amino Acid Sequence↗

PubMed1988

Fluorescence studies of phosphoribulokinase, a light-regulated, chloroplastic enzyme.

Recent characterization of spinach phosphoribulokinase has revealed that the homodimeric molecule contains only two tryptophans per 44-kDa subunit. We have performed steady-state and frequency domain studies of the intrinsic fluorescence of this protein. The fluorescence properties reflect contributions from both types of tryptophan residues. One of these appears to be relatively exposed to solvent and the quencher, acrylamide; fluoresce with a lambda max of 345 nm; decay with a fluorescence lifetime of 6.3 ns; have a relatively red-shifted absorption spectrum; and have a certain degree of independent motional freedom, with respect to the protein. The other tryptophan residue appears to be more buried; fluoresce with lambda max of 325 nm; have a lifetime of 1.7 ns; have a relatively blue-shifted absorption spectrum; and not to enjoy independent motional freedom. On comparison of phase-resolved spectral data and solute quenching data, we suggest that resonance energy transfer between the blue and red tryptophan residues may occur. We also describe the strategy of simultaneously fitting Stern-Volmer quenching data collected at two emission wavelengths.

Chloroplasts↗

PubMed1988

Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis.

The unusual chemical properties of active-site Lys-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have suggested that this residue is required for catalysis. To test this postulate Lys-329 was replaced with glycine, serine, alanine, cysteine, arginine, glutamic acid or glutamine by site-directed mutagenesis. These single amino acid substitutions do not appear to induce major conformational changes because (i) intersubunit interactions are unperturbed in that the purified mutant proteins are stable dimers like the wild-type enzyme and (ii) intrasubunit folding is normal in that the mutant proteins bind the competitive inhibitor 6-phosphogluconate with an affinity similar to that of wild-type enzyme. In contrast, all of the mutant proteins are severely deficient in carboxylase activity (less than 0.01% of wild-type) and are unable to form the exchange-inert complex, characteristic of the wild-type enzyme, with the transition-state analogue carboxyarabinitol bisphosphate. These results underscore the stringency of the requirement for a lysyl side-chain at position 329 and imply that Lys-329 is involved in catalysis, perhaps stabilizing a transition state in the overall reaction pathway.

Base Sequence↗

PubMed1987

Intersubunit location of the active site of ribulose-bisphosphate carboxylase/oxygenase as determined by in vivo hybridization of site-directed mutants.

Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50.5-kDa subunits with two substrate binding sites per molecule of dimer. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interactions, we have used a novel in vivo approach for producing heterodimers from catalytically inactive, site-directed mutants of the carboxylase. When the alleles encoding these mutant proteins are placed separately into compatible plasmids and coexpressed in the same Escherichia coli host, activity is observed at about 20% of the wild-type level. Analysis of the carboxylase purified from these cells reveals the presence of heterodimers of the two mutant proteins. This interallelic complementation demonstrates that domains from each of the subunits interact to form a shared active site.

Alleles↗

PubMed1987

Essentiality of Glu-48 of ribulose bisphosphate carboxylase/oxygenase as demonstrated by site-directed mutagenesis.

Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or ligand binding. However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme. These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role. In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.

Amino Acid Sequence↗

PubMed1987

Function of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase as examined by site-directed mutagenesis.

Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman, F.C., Milanez, S., and Lee, E.H. (1985) J. Biol. Chem. 260, 13968-13975). In attempts to clarify the role of Lys-166 of the carboxylase, we have used site-directed mutagenesis to replace this lysyl residue with glycine, alanine, serine, glutamine, arginine, cysteine, or histidine. All seven of these mutant proteins, purified by immunoaffinity chromatography, are severely deficient in carboxylase activity; the serine mutant, which is the most active, has a kcat only 0.2% that of the wild-type enzyme. Although low, the carboxylase activity displayed by some of the mutant proteins proves that Lys-166 is not required for substrate binding and argues that the detrimental effects brought about by amino acid substitutions at position 166 do not reflect gross conformational changes. As demonstrated by their ability to tightly bind a transition-state analogue (2-carboxyarabinitol 1,5-bisphosphate) in the presence of CO2 and Mg2+, some of the mutant proteins undergo the carbamylation reaction that is required for activation of the wild-type enzyme. Since Lys-166 is required neither for activation (i.e. carbamylation by CO2) nor for substrate binding, it must be essential to catalysis. When viewed within the context of previous related studies, the results of site-directed mutagenesis are entirely consistent with Lys-166 functioning as the base that initiates catalysis by abstracting the C-3 proton from ribulosebisphosphate. An alternative possibility that Lys-166 acts to stabilize a transition state in the reaction pathway cannot be rigorously excluded.

Amino Acid Sequence↗

PubMed1986

Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis.

Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.

Alanine↗

PubMed1986

Purification and characterization of ribulose-5-phosphate kinase from spinach.

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.

Amino Acids↗

PubMed1986

A reconstruction of the gene for ribulose bisphosphate carboxylase from Rhodospirillum rubrum that expresses the authentic enzyme in Escherichia coli.

Escherichia coli plasmid pRR36, which expresses Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) as a fusion protein [Nargang et al., Mol. Gen. Genet. 193 (1984) 220-224], was used to construct a new clone of the carboxylase gene (rbc) whose expression product is the wild-type enzyme. This construction entailed removing all lacZ-coding sequences and a portion of the 5'-noncoding leader of the R. rubrum rbc gene. The highest specific activity of carboxylase was observed with an expression vector which juxtaposed the trp-lac (tac) hybrid promoter with the R. rubrum ribosome binding site and the rbc structural gene. The carboxylase expressed in E. coli JM107 was purified to near homogeneity and, based on subunit Mr and specific enzymic activity, the isolated protein appeared indistinguishable from authentic ribulose bisphosphate carboxylase from R. rubrum. N-terminal sequence analyses of the cloned enzyme verified that the cloned and wild-type enzymes are the same.

Base Sequence↗

PubMed1986

Distance between two active-site lysines of ribulose bisphosphate carboxylase from Rhodospirillum rubrum.

In the absence of a three-dimensional structure of ribulose-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase(dimerizing), EC 4.1.1.39], we have probed the distance between two active-site lysyl residues (Lys-166 and Lys-329) of the Rhodospirillum rubrum enzyme with 4,4'-diisothiocyano-2,2'-disulfonate stilbene, a covalent cross-linking reagent that spans 12 A. The reagent rapidly inactivated the carboxylase, and a competitive inhibitor provided substantial protection. To remove products arising from intersubunit or intermolecular cross-linking, the inactivated enzyme was subjected to gel filtration in the presence of urea. Inspection of a tryptic digest of the isolated monomeric fraction revealed that more than half of the incorporated reagent was associated with a single peptide. This peptide was purified by gel filtration, followed by high HPLC. Compositional and sequence analyses of the purified peptide established that it was composed of two chains, encompassing positions 149-168 and 314-337 of the original protein subunit and connected by a cross-link between Lys-166 and Lys-329. Thus, the two active-site lysines of the carboxylase can be juxtaposed within 12 A, a finding that is consistent with their purported proximity to ribulose bisphosphate in the enzyme-substrate complex. The cross-link was not formed when the carboxylase was treated with the reagent either in the presence of a transition-state analogue (carboxyarabinitol bisphosphate) or in the absence of CO(2) and Mg(2+), conditions under which the enzyme exists in a deactivated form.

Journal Article↗

PubMed1985

Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase.

Trinitrobenzene sulfonate rapidly inactivates ribulosebisphosphate carboxylase/oxygenase from both spinach and Rhodospirillum rubrum. With large molar excesses of the reagent, the reactions obey pseudo-first order kinetics and the rates of inactivations are directly proportional to the concentrations of trinitrobenzene sulfonate; thus, there is no indication of reversible complexation of reagent with enzyme. Saturating levels of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduce the rates of inactivations but do not prevent them, thereby suggesting that the groups subject to arylation remain accessible in the enzyme complexed with competitive inhibitor. Characterization of tryptic digests of the inactivated enzymes reveals that Lys-166 of the R. rubrum enzyme and Lys-334 of the spinach enzyme are the only major sites of arylation. Both of these lysines have been assigned to the catalytic site by prior affinity labeling studies and are found within highly conserved regions of primary structure. As a monoanion over a wide pH range, trinitrobenzene sulfonate, for which the carboxylase lacks high affinity, can thus be used to determine the pKa values of the two active-site lysyl epsilon-amino groups. Based on the pH dependency of inactivation of the R. rubrum enzyme by trinitrobenzene sulfonate, the epsilon-amino group of Lys-166 exhibits a pKa of 7.9 and an intrinsic reactivity (ko) of 670 M-1 min-1. In analogous experiments, Lys-334 of the spinach enzyme exhibits a pKa of 9.0 and a ko of 4500 M-1 min-1. Under deactivation conditions (i.e. in the absence of CO2 and Mg2+), the pKa of Lys-334 becomes 9.8 and the ko is increased to 26,000 M-1 min-1. By comparison, the reaction of trinitrobenzene sulfonate with N-alpha-acetyl-lysine reveals a pKa of 10.8 and a ko of 1250 M-1 min-1. The spinach carboxylase, catalytically inactive as a consequence of selective arylation of Lys-334, still exhibits tight binding of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate. Therefore, Lys-334 is not required for substrate binding and may serve a role in catalysis. The unusually low pKa of Lys-166 argues that this residue is also important to catalysis rather than substrate binding.

Anions↗

PubMed1985

Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase.

Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 M-1 S-1 at 2 degrees C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethyl-maleimide. The reactive sulfhydryl groups are likely those generated by reduction of a disulfide during activation.

Adenosine Diphosphate↗