Agmatinase Activity Research Articles

Putrescine Biosynthesis from Agmatine by Arginase (TtARG) in Thermus thermophilus.

In the three domains of life, three biosynthetic pathways are known for putrescine. The first route is conversion of ornithine to putrescine by ornithine decarboxylase (ODC: SpeC), the second route is the conversion of arginine to agmatine by arginine decarboxylase (ADC: SpeA), followed by the conversion of agmatine to putrescine by agmatine ureohydrolase (AUH: SpeB), and the third route is the conversion of agmatine to N-carbamoylputrescine by agmatine deiminase (agmatine iminohydrolase, AIH), followed by the conversion of N-carbamoylputrescine to putrescine by N-carbamoylputrescine amidohydrolase (NCPAH). An extreme thermophile, Thermus thermophilus produces putrescine, although this bacterium lacks homologs for putrescine synthesizing pathways, such as ODC, AUH, AIH and NCPAH. To identify genes involved in putrescine biosynthesis in T. thermophilus, putrescine biosynthesis was examined by disruption of a predicted gene for agmatinase (agmatine ureohydrolase), or by using purified enzyme. It was found that arginase (TTHA1496) showed an agmatinase activity utilizing agmatine as a substrate. These results indicate that this bacterium can use arginase for putrescine biosynthesis. Arginase is a major contributor to putrescine biosynthesis under physiological conditions. The presence of an alternative pathway for converting agmatine into putrescine is functionally important for polyamine metabolism supporting survival at extreme environments.

Enzymatic characterization of agmatinase (AGM-1) from the filamentous fungus Neurospora crassa

Agmatinase is a metallohydrolase involved in the hydrolysis of agmatine to produce urea and putrescine. Although its role in organisms is still under study, there are no reports of this family of enzymes in filamentous fungi. Recently, a protein showing agmatinase activity was reported in Neurospora crassa. Therefore, the aim of this work is to determine if the protein (AGM-1) found in the filamentous fungus N. crassa is a true agmatinase. The protein AGM-1was purified directly from N. crassa cultures, and its enzymatic characterization was carried out. The catalytic parameters such as optimum pH, thermostability, transformation kinetics, and activity in the presence of a cofactor were determined. The results show that AGM-1 can use manganese as a cofactor for its enzymatic activity, showing a transformation rate constant (kcat) of 77 s−1 and an affinity constant (KM) of 50.5 mM. The protein loses 50% of its activity when incubated 15 min at 30 °C and reaches maximal enzymatic activity at a pH range of 8–8.5. Our results indicate that the AGM-1 from N. crassa shows similar characteristics to true agmatinases already reported in other organisms. Thus, our findings strongly support that the protein annotated as hypothetical agmatinase in N. crassa is a true agmatinase.

Insights into the Mn2+ Binding Site in the Agmatinase-Like Protein (ALP): A Critical Enzyme for the Regulation of Agmatine Levels in Mammals.

Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn2+ for catalytic activity. However, the Mn2+ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn2+ binding site of the ALP and we propose new putative Mn2+ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the “central-ALP”, which include the putative ligands of Mn2+. The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered Km for agmatine and a decreased kcat. Similar to wild-type ALP, central-ALP is activated by Mn2+ with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn2+ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.

The virulence factor urease and its unexplored role in the metabolism of Cryptococcus neoformans.

Cryptococcal urease is believed to be important for the degradation of exogenous urea that the yeast encounters both in its natural environment and within the human host. Endogenous urea produced by the yeast's own metabolic reactions, however, may also serve as a substrate for the urease enzyme. Using wild-type, urease-deletion mutant and urease-reconstituted strains of Cryptococcus neoformans H99, we studied reactions located up- and downstream from endogenous urea. We demonstrated that urease is important for cryptococcal growth and that, compared to nutrient-rich conditions at 26°C, urease activity is higher under nutrient-limited conditions at 37°C. Compared to cells with a functional urease enzyme, urease-deficient cells had significantly higher intracellular urea levels and also showed more arginase activity, which may act as a potential source of endogenous urea. Metabolic reactions linked to arginase were also affected, since urease-positive and urease-negative cells differed with respect to agmatinase activity, polyamine synthesis, and intracellular levels of proline and reactive oxygen species. Lastly, urease-deficient cells showed higher melanin levels at 26°C than wild-type cells, while the inverse was observed at 37°C. These results suggest that cryptococcal urease is associated with the functioning of key metabolic pathways within the yeast cell.

The Role of Reduced Polyamine Synthesis in Ischemic Stroke

Our aim was to determine the levels of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and agmatine, ornithine, nitric oxide (NO) and arginase activity in the acute and subacute phases of acute ischemic stroke (AIS), by revealing the possible role of these levels in neuroinflammation and neurogenesis that are postulated to begin after AIS. 35 patients diagnosed with AIS and 35 controls were included in the study. ADC, ODC, agmatinase and NO levels were determined by ELISA kits. Arginase activity and ornithine levels were measured spectrophotometrically. There was no statistically significant difference between the levels of agmatinase, ornithine and arginase activity on days 1 and 7 in the AIS group and the levels measured in the control group (p > 0.05). High ADC and low ODC and NO values were found in the patients compared to the controls in both acute and subacute periods (p < 0.05). It was shown that polyamine synthesis decreased in both acute and subacute periods after AIS; and this was due to the decrease in ODC levels, the rate-regulating step of the major pathway. This reduction may be associated with both the inadequate stimulation of neurogenesis and the immune system with reduced NO synthesis. polyamine synthesis enzymes, ornithine, ornitine decarboxylase, nitric oxide, arginine decarboxylase, agmatine, agmatinase, arginase activity, neuroinflammation, neurogenesis, immune system, acute ischemic stroke.

Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction

Ureohydrolases are members of the metallohydrolase family of enzymes. Here, a simple continuous assay for agmatinase (AGM) activity was established by following the degradation of agmatine to urea and putrescine using isothermal titration calorimetry (ITC). ITC is particularly useful for kinetic assays when substrates of interest do not possess suitable chromophores that facilitate the continuous spectrophotometric detection of substrate depletion and/or product formation. In order to assess the accuracy of the ITC-based assay, catalytic parameters were also determined using a discontinuous, colorimetric assay. Both methods resulted in comparable kinetic parameters. From the colorimetric assay the kcat and KM values are 131 s−1 and 0.25 mM, respectively, and from the ITC assay the corresponding parameters are 30 s−1 and 0.45 mM, respectively. The continuous ITC-based assay will facilitate functional studies for an enzyme that is an emerging target for the development of addiction treatments.

Identification of agmatinase from Vibrio splendidus and its roles in modulating arginine metabolism of Apostichopus japonicus

Innate immunity is the first line of defense against invading pathogens. However, pathogenic bacteria have developed various mechanisms, such as direct competition for common substrates and transformation of metabolic regulations of the host, which allow them to escape from the immune surveillance system and enhance their survival. In this study, the Vibrio splendidus agmatinase (designated as Vsagmatinase) gene was cloned, and its biological activities in Apostichopus japonicus coelomocytes were analyzed. Vsagmatinase encoded an open reading frame of 930 bp, which translated into a predicted protein of 309 amino acid residues. SMART analysis and multiple sequence alignment indicated that Vsagmatinase displayed an arginase domain, and the conserved histidine (H151 and H163) and aspartic acid (D125, D149, D153, D231, and D233) residues for catalytic activity were all strictly conserved in this domain. Moreover, four conserved residues, namely, Asp153, His163, Thr245, and Glu275, that bonded with the guanidinium portion of agmatine were conserved in V. splendidus agmatinase. The recombinant protein was expressed in Escherichia coli (BL21), and the purified Vsagmatinase showed high agmatinase activity with agmatine substrate. By site-directed mutagenesis, the agmatinase activity of mutated Vsagmatinase was reduced to approximately 8.9%–9.4% of wild-type activity under different Vsagmatinase protein concentrations, indicating that the Glu275 residue was necessary for the guanidinium group of agmatine. Notably, injection of Vsagmatinase in coelomocytes could significantly increase both the mRNA expression levels and enzyme activities of Ajarginase and Ajagmatinase and obviously inhibited AjNOS transcript and NO content in A. japonicus. By contrast, mutated Vsagmatinase downregulated the activity of arginase and promoted NO production at 12 h after injection. Our findings suggested that Vsagmatinase served as the competitor that utilizes host agmatine and modulates arginine metabolism in A. japonicus.

The Involvement of Agmatine in Individual Pain Sensitivity

Distribution of agmatinase (AGM), an enzyme of agmatine metabolism, was studied in the spinal cord and dorsal root ganglia of rats with different levels of initial thermal pain sensitivity under normal conditions and during the development of inflammatory pain using immunohistochemistry. The initial type of pain threshold affects the specific pain response of an animal to pain so that the rats with a higher threshold exhibit a less-expressed behavioral pain response. We found a specific distribution and time course of activity of AGM in the brain regions of animals with different nociceptive thresholds. The animals with a lower threshold had a higher number of AGM-immunoreactive neurons in the ganglia and a lower density of fibers and neurons in the dorsal roots of the spinal cord compared to the animals with a higher threshold. The development of tonic pain was associated with differently directed changes of AGM activity in the studied structures and depended on the threshold of nociception. Our data suggest that the endogenous system of agmatine metabolism may be involved in the initial threshold of nociception, as well as in organization of the behavioral response to pain.

Cloning of two LIMCH1 isoforms: characterization of their distribution in rat brain and their agmatinase activity.

Agmatine, a precursor for polyamine biosynthesis, is also associated with neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. This molecule results from the decarboxylation of L-arginine by arginine decarboxylase, and it is hydrolyzed to urea and putrescine by agmatinase. Recently, we have described a new protein that also hydrolyzes agmatine, agmatinase-like protein (ALP), which was identified through immunohistochemical analysis in the hypothalamus and hippocampus of rats. However, its sequence differs greatly from all known agmatinases and does not contain the typical Mn(2+) ligands associated with the urea hydrolase family of proteins. ALP has a LIM-like domain close to its carboxyl terminus, and the removal of which results in a truncated variant with a tenfold increased k cat value and a threefold decreased K m value for agmatine. Analysis of the gene database revealed several transcripts, denominated LIMCH1 isoforms, with extreme 3' sequences identical to ALP. Limch1 gene products have been described as members of a multi-domain family of proteins with the biggest isoform containing a calponin homology (CH) domain at its N-terminus. Here, we cloned two LIMCH1 transcripts, one of 3177bp and the other of 2709bp (ALP contains 1569bp) and analyzed LIMCH1 expression and distribution in rat brain using RT-PCR, Western blot and immunohistochemical analyses. LIMCH1 was detected mainly in the hypothalamic and hippocampal regions, which is similar to the distribution of ALP and agmatine in brain. In addition, we cloned and expressed both isoforms in E. coli and confirmed that they were catalytically active on agmatine with kinetic parameters similar to ALP. LIM domain-truncated variants of both isoforms moderately increased the k cat and catalytic efficiency. Thus, we propose that LIMCH1 is useful to regulate the intracellular concentrations of the neurotransmitter/neuromodulator, agmatine.

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase.

Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on l-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome.

Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [14C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The Δalr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [14C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the Δalr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

Expression and localization of an agmatinase-like protein in the rat brain

Agmatinase catalyzes the hydrolysis of agmatine into putrescine and urea, and agmatine (decarboxylated L: -arginine) plays several roles in mammalian tissues, including neurotransmitter/neuromodulatory actions in the brain. Injection of agmatine in animals produces anticonvulsant, antineurotoxic and antidepressant-like actions. Information regarding the enzymatic aspects of agmatine metabolism in mammals, especially related to its degradation, is relatively scarce. The explanation for this is the lack of enzymatically active preparations of mammalian agmatinase. Recently, we have cloned a protein from a cDNA rat brain library having agmatinase activity although its amino acid sequence greatly differs from all known agmatinases, we called agmatinase-like protein. In this work, we analyzed the expression of this enzyme in the rat brain by means of RT-PCR and immunohistochemical analysis using a polyclonal antibody generated against the recombinant agmatinase-like protein. The agmatinase-like protein was detected in the hypothalamus in glial cells and arcuate nucleus neurons, and in hippocampus astrocytes and neurons, but not in brain cortex. In general, detected localization of agmatinase-like protein coincides with that described for its substrate agmatine and our results help to explain several reported effects of agmatine in the brain. Concretely, a role in the regulation of intracellular concentrations of the neurotransmitter/neuromodulator agmatine is suggested for the brain agmatinase-like protein.

Biochemical and immunohistochemical analysis of an unusual rat brain agmatinase

Agmatine plays neurotransmitter/neuromodulatory actions in the brain. It result from decarboxylation of arginine by arginine decarboxylase and it is hydrolysed by agmatinase. Recently, we have cloned an unusual rat brain protein with agmatinase activity, although their deduced sequences greatly differ from all known agmatinases. However, in spite of only 12% identity with E. coli agmatinase, the rat brain protein is recognized by a polyclonal antibody raised against the bacterial enzyme. Like E. coli agmatinase, the rat brain enzyme exhibits an absolute requirement for Mn2+, not related to a metal‐binding LIM domain detected in its sequence. As shown here, the Mn2+ requirement is retained by a variant lacking the domain. We, therefore, associate Mn2+ to the hydrolytic reaction in the enzyme active site. Also described here is the generation of a polyclonal antibody against the brain agmatinase, which also reacts with the E. coli enzyme. We conclude that, although not sequence‐related, the rat brain agmatinase is structurally related to all known agmatinases. By immunohistochemical analysis, the enzyme was detected in arquate nucleus, ciliated ependymal cells and tanycytes hypothalamic. Since this coincide with that for agmatine, the enzyme would play a central role in regulating the levels and thus, the neurotransmitter/neumoregulatory actions of the primary amine in the brain. Grant FONDECYT 11070069

Cloning and functional expression of a rodent brain cDNA encoding a novel protein with agmatinase activity, but not belonging to the arginase family

A rat brain cDNA encoding for a novel protein with agmatinase activity was cloned and functionally expressed. The protein was expressed as a histidine-tagged fusion product with a molecular weight of about 63 kDa. Agmatine hydrolysis was strictly dependent on Mn 2+; K m and k cat values were 2.5 ± 0.2 mM and 0.8 ± 0.2 s −1, respectively. The product putrescine was a linear competitive inhibitor ( K i = 5 ± 0.5 mM). The substrate specificity, metal ion requirement and pH optimum (9.5) coincide with those reported for Escherichia coli agmatinase, the best characterized of the agmatinases. However, as indicated by the k cat/ K m (320 M −1 s −1), the recombinant protein was about 290-fold less efficient than the bacterial enzyme. The deduced amino sequence revealed great differences with all known agmatinases, thus excluding the protein from the arginase family. It was, however, highly identical (>85%) to the predicted sequences for fragments of hypothetical or unnamed LIM domain-containing proteins. As a suggestion, the agmatinase activity is adscribed to a protein with an active site that promiscuously catalyze a reaction other than the one it evolved to catalyze.

Mutational analysis of substrate recognition by human arginase type I − agmatinase activity of the N130D variant

Upon mutation of Asn130 to aspartate, the catalytic activity of human arginase I was reduced to approximately 17% of wild-type activity, the Km value for arginine was increased approximately 9-fold, and the kcat/Km value was reduced approximately 50-fold. The kinetic properties were much less affected by replacement of Asn130 with glutamine. In contrast with the wild-type and N130Q enzymes, the N130D variant was active not only on arginine but also on its decarboxylated derivative, agmatine. Moreover, it exhibited no preferential substrate specificity for arginine over agmatine (kcat/Km values of 2.48 x 10(3) M(-1) x s(-1) and 2.14 x 10(3) M(-1) x s(-1), respectively). After dialysis against EDTA and assay in the absence of added Mn2+, the N130D mutant enzyme was inactive, whereas about 50% full activity was expressed by the wild-type and N130Q variants. Mutations were not accompanied by changes in the tryptophan fluorescence properties, thermal stability or chromatographic behavior of the enzyme. An active site conformational change is proposed as an explanation for the altered substrate specificity and low catalytic efficiency of the N130D variant.

Insights into the interaction of human arginase II with substrate and manganese ions by site‐directed mutagenesis and kinetic studies

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.

N1-Aminopropylagmatine, a New Polyamine Produced as a Key Intermediate in Polyamine Biosynthesis of an Extreme Thermophile, Thermus thermophilus

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.

Effect of chronic morphine treatment on the biosynthesis of agmatine in rat brain and other tissues

Agmatine is an endogenous amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC), and metabolized to putrescine by agmatinase. Exogenously administered agmatine has several biological actions including its ability to potentiate morphine analgesia and block symptoms of morphine tolerance/withdrawal in rats. To investigate the role of endogenous agmatine in this action, we sought to determine whether chronic exposure to morphine and induction of withdrawal modulate the synthesis of agmatine in rat brain and other tissues. Exposure of rats to morphine for three days significantly decreases the activity of ADC and the levels of agmatine in rat liver, kidney, brain, aorta and intestine with no changes in agmatinase activity. The precipitation of withdrawal syndrome by injecting naloxone further decreases ADC activity and agmatine levels in these tissues. We conclude that endogenous agmatine may play an important role in regulating morphine tolerance/dependence and withdrawal symptoms.

Evidence That Histidine-163 Is Critical for Catalytic Activity, but Not for Substrate Binding to Escherichia coli Agmatinase

Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme–protector complex (12 mM) was essentially equal to the Ki value for this compound acting as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3–5% of wild-type activity, without any change in Km for agmatine or Ki for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivations. We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding.

Manganese Is Essential for Catalytic Activity ofEscherichia coliAgmatinase

PurifiedEscherichia coliagmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+(0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 ± 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.