314
Agents and Actions vol. 9/4 (1979) Birkh/iuser Verlag, Basel
Peptide Inhibition of Mammalian Histidine Decarboxylase I by
LENA HAMMAR a n d ULF RAGNARSSON
Department of Biochemistry, Biomedical Center, University of Uppsala, Box 576, S-751 23 Uppsala, Sweden
Abstract The hypothesis that N-terminal hlstidlne peptides might act as inhibitors to histldine deearboxylase was investigated. A murine mastoeytoma was utilized as enzyme source. The crude extract of this tissue exhibits high rates of deearboxylation of both histidine and DOPA and was used to establish the specificity in the effect of the compounds tested. For kinetic analyses a highly purified hlstidine deearboxylase fraction was used. The effect of some representative peptides on both enzyme activities were recorded. Histidine decarboxylase exclusively was inhibited by N-termlnal histidine peptides. None of the other peptides investigated interfered negatively with this enzyme. This inhibition was consistent in the purified preparation and appeared to be more pronounced with increasing hydrophobieity in the second amino acid. Histidyl-phenylalanine was found to be about 100-fold as potent as the commonly used specific histldine deearboxylase inhibitor a-methyl histidine. It is concluded that small peptides with histidine as the N-termlnai amino acid might act as specific inhibitors for mammalian hlstidlne deearboxylase. An analog effect of small tyrosyl or phenylalanyl peptldes was not seen for the DOPA deearboxylase.
Introduction The biosynthesis of histamine proceeds via decarboxylation of histidine. The mammalian enzymes found to catalyse this reaction might differ in specificity. Histidine decarboxylase (EC 4.1.1.22) is characterized by a pH optimum at or below neutrality while an obviously different and less specific enzyme decarboxylates histidine at pH 8 or above [I-4, among others]. The latter enzyme might be identical with aromatic L-amino acid decarboxylase (EC 4.1.1.28) since its activity is blocked by the aromatic amino acid decarboxylase inhibitor orJ Presented in part at the 1 lth FEBS Meeting, Copenhagen, August 1977.
methyl DOPA [5, further refs. in 2]. Purified aromatic amino acid decarboxylase have been reported to, besides its preferred substrates DOPA and 5-hydroxy tryptophan, also to some extent act on histidine [6] but there are also contradictory results [4, 7, 8]. In attempts to find inhibitors specific for mammalian histidine decarboxylase (EC 4.1.1.22) different lines have been followed. Several imidazole and histidine analogs have been tried as potential histidine decarboxylase inhibitors [5, 9-16]. From these reports one might conclude that (i) substitution in the imidazole-ring is not allowed [9, I0], (it')/1-substitution is not allowed [I0, 1 I]. (iii) Substitution of a methyl group for the or-hydrogen yields an apparently specific inhibitor, or-methyl histidine (a, Scheme 1). An ethyl or higher alkyl group in the or-position gives compounds with less or no inhibitory effect on histidine decarboxylase [9, 11]. (iv) A free amino group is preferred, but might not be essential [9, 13], and (v) substitution at the carboxyl group in histidine has given some working inhibitors, among them the strongest reported among the histidine analogs; 4imidazolyl-3-amino-2-butanone [14, 15] (b, Scheme 1) and histidine methylester [9, 13] (c, Scheme 1). Apart from the histidine analogs, some benzoic acid derivatives [17, 18] and substituted phenols ]181, (+)Catechin [19[, some rhodanines [20] and methoxyamines [21-231 have been described as inhibitors for histidine decarboxylase. The methoxyamines, such as Brocresine (NSD 1055), are the strongest inhibitors known for the enzyme but are not specific for histidine decarboxylase. They are aldehydic reagents
315
Peptide Inhibition of Mammalian Histidine Decarboxylase
J7% ~ HIS
.."x
%,
17"'/0 I t \c.. (hi
~ (o1
HN--~~ 0 ["'N 1~)' O_--,CH3)3CH 3
To establish the specificity of their action comparison is made with their effects on aromatic L-amino acid decarboxylase, assayed as DOPA decarboxylase. The inhibitors a-methyl histidine and a-methyl DOPA of the respective decarboxylases, as well as several non-histidyldipeptides are included as controls.
Materials and methods
Enzymes
which apparently bind to the aldehyde group of the enzyme-bound pyridoxal phosphate, and therefore interferes with other pyridoxal phosphate-dependent enzymes as well. However, some selectivity has been observed with different methoxyamines. HUSZTI et al. [22] for instance, report that 3-nitrobenzyloxyamine and pyridyl-3methoxyamine have about 20-fold higher affinity for histidine decarboxylase than for aromatic Lamino acid decarboxylase, while some other benzyloxyamines and pyridylmethoxyamines were about equally potent with the two enzymes. The cited reports on histidine analog inhibition suggest that the substrate binding site is a narrow pocket highly specific for hlstidine. There is apparently some leeway in the catalytic site around the a-carbon, but only in the direction distal to histidine carbon-1 is there reasonable steric freedom as judged from the fact that alkylesters up to butyl are inhibitory [9]. The strong inhibition observed with benzyl and pyridyl substituted methoxyamines [22] suggests a hydrophobic area close to the catalytic center. Against this background it would seem likely that peptides with histidine at the amino terminus could act as inhibitors (d, Scheme 1). AURES and CLARK [24] found that histidyl-histidine did inhibit histidine decarboxylation in mouse mastocytoma to about the same extent as did ~methyl histidine but KELLEY et al. [9] tried histidylglycine as inhibitor to the rat stomach enzyme and found no effect at a peptide concentration of 0.1 raM. However, we have earlier noted a weak but apparently specific inhibition of histidine d e c a r b o x y l a s e b y histidyl-lysine a n d a histidylp e n t a p e p t i d e [25]. T h e p r e s e n t p a p e r c o n c e r n s the action o f several small histidyl-peptides o n the activity o f a m a m m a l i a n histidine d e c a r b o x y l a s e .
A crude extract of histidine and DOPA decarboxylase was prepared from a transplantable mast cell tumour of mice [26]; homogenization under argon was followed by centrifugation (150,000 x g, 2 h) and dialysis against 0.15 M potassium phosphate, 10 IJM pyridoxal phosphate, 1 mM dithiothreitol, 3 mM NaNj (pH 6.8). The specific activity of histidine decarboxylase in this crude extract was about 9 nmoles CO2/h.mg protein and the specific activity of DOPA decarboxylase was about 6 nmoles CO2/h.mg protein. Purification by hydroxyapatite chromatography, molecular sieving and chromatography on octyl Sepharose (to be published elsewhere) yielded a histidine decarboxylase with the specific activity of 1200 nmoles CO2/h.mg protein (the coefficient of variation in 8 different activity determinations was 9%). Only traces of DOPA decarboxylase activity was found in this partially purified preparation, which was used for the kinetic analysis of the effect of histidyl-phenylalanine on histidine decarboxylase.
Assay Decarboxylase activity was assayed as the amount ot 14CO2 released by the enzyme extract from (1-14C)-sub. strates at pH 6.8 and 37~ under argon during 1 h (DOPA decarboxylase) or 2 h (histidine decarboxylase) [261. The reaction mixture in a total volume of 50/J1 contained enzyme extract in amount to give an output in the histidinc decarboxylase controls (no inhibitor added) when assayed al 0.8 mM histidine of about 1300 cpm (1.2 nmoles CO2/h ) ir the case of the crude enzyme and about 2500 cpm (2.2 nmoles CO2/h) in the case of the purified enzyme (for th~ kinetic plot half this amount), L-histidine or L-DOPA, th( substance to be tested, 10 tJM pyridoxal phosphate, 1 m& dithiothreitol, 3 mM NAN3, 0.15 M potassium phosphat, (pH 6.8) and 40 nCi 1J4C-substrate. To minimize the erro: in the low-substrate activity measurement in the kineti, analyses the concentration of the substrate was varied whih the amount of radioactivity was kept constant. Thus th, blank level was kept within 30-50 cpm. The substrat~ concentration in the kinetic analysis was varied in the rang, 8-808 ~tM.
Chemicals The histidyl-dipeptides were from Bachem, Bubendor Schweiz. Their purity was confirmed by amino acid analys: after acid hydrolysis. The His. Arg. Ala. Ser. Val was syz thesized for other purposes and was available in the lab. L- i 14C-histidine and DL-I-~4C-DOPA were from New Englan Nuclear.
316
Peptide Inhibition of Mammalian Histidine Decarboxylase
Results
The effect of some small peptldes on the activity of histldine decarboxylase and DOPA decarboxylase Histidine decarboxylase and DOPA decarboxylase were assayed at saturating substrate concentrations (0.80 m M and 0.30 mM, respectively) in the presence of the compounds listed in Table 1. The results of the experiments with histidine decarboxylase are given in Table 1 and show that the histidine decarboxylase activity of both the crude extract and the purified enzyme is inhibited by histidyl-containing peptides. The non-histidylpeptides tested were not inhibitory. Among the Table 1
The effect of some small peptides, phenylalanine, a-methyl histidine and a-methyl D O P A on the activity of histidine decarboxylase in crude extract and in a partly purified preparation. Additive
Relative activity a as % of control with no additive Crude extract
compounds tested here the most effective inhibitor was the dipeptide His. Phe, closely followed by His. Tyr. Phenylalanine alone or in dipeptides lacking histidine had no effect. Since DOPA decarboxylase can under certain conditions decarboxylate histidine [6] it was of interest to determine whether this enzyme was also affected by the histidyl-dipeptides. In the presence of 0.5 m M c~-methyl DOPA the DOPA decarboxylase activity decreased to 42% of controls, but no significant effect was observed with any of the peptides listed or with 4 m M ~t-methyl histidine. Kinetics of the histidyl-phenylalanine inhibition of hlstldine decarboxylase The inhibition by histidyl-phenylalanine was further studied using the partially purified histidine decarboxylase. In a double reciprocal plot of kinetic data (Fig. 1) the lines intersect to the h,mg I nmol C02 /
Purified enzyme 31 ~
Concentration of additive: HIS. P H E HIS.TYR HIS. A L A HIS. HIS HIS.SER a-Methyl HIS HIS. LYS HIS. ASP HIS. A R G . A L A . SER. VAL LYS. VAL VAL. A L A GLY- LEU ALA. ALA ALA. GLY LYS.PHE PHE. VAL PHE. PHE e TRP. PHE e TYR. PHE c PHE a-Methyl D O P A
4 mM
0.2 m M
24
4 mM 3b 4b 23 b 25 b 39 b 40 b 42 b 79 b
25 22 31 40 27 32
0.8 m M
~ ....
I
/
100 I 200
12 b 19 b 52 b 64 b 75 b
~'00 ~ M / / /.DO
89 86 2O0
51 103 100 106 106 120 101 103
103
117
95
108 104 113 107 100
5
10
15
20
25 mM "1
1
100
a The average of four or more determinations are given. The standard error of the mean was below 5% of the given values except in the case of HIS. ALA, 0.8 mM, when it was 9%. b The lower limit of the normal distribution of the purified controls (n = 8) was 79% P < 0.01 and 73% P < 0.001. c Higher concentrations than 0.2 m M could not be tested due to the limited solubility of these peptides.
Figure 1 Double reciprocal kinetic plot of partially purified histidine decarboxylase at different concentrations of histidyl-phenyl-
alanine. Inserted: The slope and the 1IV intercepts of the lines from the double reciprocal plot vs. concentration of the peptide inhibitor. The lines are adopted by least square analysis to data obtained from triplicate determinations at five substrate concentrations (808, 178, 88, 38 and 8/zM, the last not shown in the figure) and the concentration of histidyl-phenylalanine in #molar as indicated on the lines.
317
Peptide Inhibition of Mammalian Histidine Decarboxylase
left of the 1/V axis, indicating that the inhibition might not be purely competitive. Secondary plots gives K~I~ = 44 # M and K~ntereept -- 244 #M. The apparent K m derived from the primary plot was 305 #M which is in agreement with what has been reported for the enzyme from the same and other sources when assayed under similar conditions [1-4, 27, 28]. All lines were derived by regression analysis. The regression coefficients are 0.98 and above, except in the case of the secondary plot of the 1/V intercepts where the regression coefficient was 0.96.
Discussion The concept that small peptides with the substrate amino acid as N-terminal might act as inhibitors for amino acid decarboxylases has not received much attention earlier. Our positive results for mammalian histldine decarboxylase might not hold for other enzymes. Two phenylalanyl, tryptophanyl and a tyrosyl dipeptide failed to inhibit the DOPA decarboxylase in our preparations. The inhibitory potency of the histidyldipeptides investigated apparently increases with increasing hydrophobicity in the second amino acid. This is consistent with the observation by MOLE et al. [18] that increasing hydrophobicity in m-substituted, benzoic acid derivatives correlates with increased inhibitory potency toward histidine decarboxylase. Comparison of our data with those given for the effect of histidine esters on rat stomach histidine decarboxylase [9] implies that the methylester is a stronger inhibitor than the histidyl-phenylalanine. The K I given for the histidine methylester was 1.8 #M, as compared to 44 #M found here for the histidyl-phenylalanine. However, the data might not be directly comparable since the enzyme sources differ and we needed almost a 10-fold higher concentration of the a-methyl histidine to get a relative inhibition of the mastocytomal enzyme comparable to that observed for the rat stomach enzyme. It is assumed that the histidine decarboxylation assayed is performed by the specific histidine decarboxylase (EC 4.1.1.22), since a-methyl DOPA had no effect on the observed histidine decarboxylation. Further evidence for this assumption is that the inhibition of histidine decarboxylation observed by a-methyl histidine and the histidyl-peptides is consistent in the purified preparation, where the DOPA de-
carboxylase activity is negligible compared to the histidine decarboxylase activity. The lack of effect of the histidyl-peptides on DOPA decarboxylase activity therefore strongly suggests that the inhibition observed is specific for the histidine decarboxylase. Small peptides in brain and other tissues have been shown to be regulatory. It is tempting to suggest that natural histidyl-peptides might operate on histidine decarboxylase in the regulation of histamine formation. Acknowledgments This work was supported by grants from the Finsen Foundation, Stockholm and Faculty of Science, Uppsala. The skilful technical assistance of Miss B. Ahlstrom is thankfully acknowledged. Received 15 January 1979
References [ 1] D. AURES and R. HAKANSSON,Histidine Decarboxylase (Mammalian), in: Methods in Enzymology, vol. XVII, part B (Eds. H. Tabor and G.W. Tabor; Academic Press, New York and London, 1970), pp. 667-677. [2] R. HAKANSSON.New Aspects of the Formation and Function of Histamine, 5-Hydroxytryptamine and Dopamine in Gastric Mucosa, Acta Physiol. Stand. Suppl. 340. 3-134 (1970). [3] H. WEISSBACH,W. LOVENaERGand S. UDENERIEND, Characteristics of Mammalian Histidine Decarboxylating Enzymes, Biochim. Biophys. Acta 50, 177-179 (1961). [4] W. LORENZ, ST. HALBACH, M. GERANT and E. WERLE, Specific Histidine Decarboxylases in the Gastric Mucosa of Man and Other Mammals. Determination, Location and Properties, Biochem. Pharmac. 18, 2625-2637 (1969). [5] B. ROBINSONand D.M. SHEPHARD,The Inhibition of the L-Histidine Decarboxylases of Guinea-Pig Kidney and Rat Hepatoma, J. Pharm. Pharmac. 14, 9-15 (1962). [6] J.G. CHRISTENSON,W. DAIRMANand S. UDENERIEND, Preparation and Properties of a Homogeneous Aromatic L-Amino Acid Decarboxylase from Hog Kidney, Arch. Biochem. Biophys. 141, 356-367 (1970). [7] E. WERLE and D. AURES, Uber die Reinigung und Spezifitdt der DOPA-Decarboxylase, Hoppe-Seyler's Z. Physiol. Chem. 316, 45-60 (1959). [8] J. AWAPARA, R.P. SANDMAN and C. HANLY, Activation of DOPA Decarboxylase by Pyridoxal Phosphate, Arch. Biochem. Biophys. 98, 520-525 (1962). [9] J.L. KELLY, C.A. MILLER and H.L. WHITE, Inhibition of Histidine Decarboxylase. Derivatives of Histidine, J. Med. Chem. 20, 506-509 (1977). [101 J.L. KELLY, C.A. MILLER and E.W. MCLEAN, Attempted Inhibition of Histidine Decarboxylase with fl-Alkyl Analogues of Histidine, J. Med. Chem. 20, 721-723 (1977). [11] J.I. DEGRAw, J. ENGSTROM,M. ELLIS and H.L. JOHNSON, Potential Histidine Deearboxylase
318
[12] [131
[141
[15]
[16]
[17]
[18]
[19]
Peptide Inhibition of Mammalian Histidine Decarboxylase
lnhibitors. 1. c~and fl-Substituted Histidine Analogues, J. Med. Chem. 20, 1671-1673 (1977). B. ROBINSON and D.M. SHEPHARD, Inhibition of Histidine Decarboxylases, Biochem. Biophys. Acta 53, 431-433 (1961). E.E. SMISSMAN and J.A. WEIS, Specificity in Enzyme Inhibition. 1. Synthesis of 4-(4-Imidazolyl)-3-amino-2butanone, 4-(4-Imidazolyl)-3-acetamido-2-butanone, and 4-(4-1midazolylmethyl)-2,5-dimethyloxazole for Assay as lnhibitors of Histidine Decarboxylase, J. Med. Chem. 14, 945-947 (1971). E.E. SMISSMAN and V.D. WARNER, Specificity in Enzyme Inhibition. 2. cr Acids as Inhibitors of Histidine Decarboxylase and 3,4Dihydroxyphenylalanine Deearboxylase, J. Med. Chem. 15, 681-682 (1972). R.J. TAYLOR, JR., F.J. LEINWEBERand G.A. BRAUN,4Imidazolyl-3-amino-2-butanone (MeN-A-1293), a New Specific Inhibitor of Histidine Decarboxylase, Biochem. Pharmac. 2, 2299-2310 (1973). J.I. DEGRAw, J.S. ENGSTROM, M. ELLIS and H.L. JOHNSON, Potential Histidine Decarboxylase Inhibitors. II. 3-(4-Imidazolyl)-2-pyridine and Piper# dinecarboxylates, J. Heterocyclic Chem. 15, 217-219 (1978). H. UMEZAWA, N. SHIBAMOTO, H. NAGANAWA, S. AYUKAWA, M. MATSUZAKI, K. KONO and T. SAKAMOTO, Isolation of Lecanoric Acid, an Inhibitor of Histidine Decarboxylase from a Fungus, J. Antibiotics 27, 587-596 (1974). K.H. MOLE, D.M. SHEPHARD and J. M. WATKINS, Stmcture-action Relationships of Histidine Deearboxylase Inhibitors, Archs Int. Pharmacodyn. Th6r. 216, 192-196 (1975). H.-J. REIMANN, W. LORENZ, M. FISCHER, R. FROLICH and H.-J. MEYER, Histamine and Acute Haemorrhagic Lesions in Rat Gastric Mucosa: Prevention of Stress Ulcer Formation by ( +)-Catechin, an Inhibitor of Specific Histidine Decarboxylase in vitro, Agents and Actions 7, 69-74 (1977).
[20} C.A. FREE, E. MAJCHROWICZ and S.M. HESS, Mechanism of Inhibition of Histidine Decarboxylase by Rhodanines, Biochem. Pharmac. 20, 1421-1428 (1971). [21] Z. HUSZTI, E. KASZTREINER, M. KORTI, M. FEKETE and J. BORSY, 2-hydroxy-5-Carbomethoxybenzyloxyamine: A new Potent Inhibitor of Histidine Deearboxylase, Biochem. Pharmac. 22, 2253-2265 (1973). [22] Z. HUSZTI, E. KASZTREINER, G. SZILAGYI,J. KOSARY and J. BORSY, Decarboxylase Inhibition and Structure-Activity Relationship Studies with some Newly Synthetized Benzyloxyamine and Pyridylmethoxyamine Derivatives, Biochem. Pharmac. 22, 2267-2275 (1973). [23l L. ELLENBOGEN, R.G. KELLY, R.J. TAYLOR, JR. and C.S. STUBBS,JR., Studies on the Inhibition of Histidine Decarboxy lase, A romatic- L-A mino Acid Decarboxy lase and Acid Secretion by Brocresine and its Metabolltes, Biochem. Pharmac. 22, 939-947 (1973). [24] D. AURES and W.G. CLARK,A Rotating Diffusion Chamber for C1402 Determination as Applied to Inhibitor Studies on Mouse Mast Cell Tumor Histidine Decarboxylase, Anal. Biochem. 9, 35-47 (1964). [251 L. HAMMAR, Studies on Mammalian Histidine Decarboxylase, Acta Universitatis Upsaliensis, 398 (1977). [26] L. HAMMAR, S. PAHLMANand S. HJERTEN, Chromatographic Purification of a Mammalian Histidine Decarboxylase on Charged and Non-Charged Alkyl Derivatives of Agarose, Biochim. Biophys. Acta 403, 554-562 (1975). [27] W.W. NOEL, Some Properties of Mouse Mastocytoma Histidine Decarboxylase, Thesis, Yale University School of Medicine, April, 1965. [28] D.G. RITCHIE and D.A. LEVY, A Microassay for Mammalian Histidine Decarboxylase, Anal. Biochem. 66, 194-205 (1975).
Agents and Actions vol. 9/4 (1979) Birkh/iuser Verlag, Basel
Peptide Inhibition of Mammalian Histidine Decarboxylase I by
LENA HAMMAR a n d ULF RAGNARSSON
Department of Biochemistry, Biomedical Center, University of Uppsala, Box 576, S-751 23 Uppsala, Sweden
Abstract The hypothesis that N-terminal hlstidlne peptides might act as inhibitors to histldine deearboxylase was investigated. A murine mastoeytoma was utilized as enzyme source. The crude extract of this tissue exhibits high rates of deearboxylation of both histidine and DOPA and was used to establish the specificity in the effect of the compounds tested. For kinetic analyses a highly purified hlstidine deearboxylase fraction was used. The effect of some representative peptides on both enzyme activities were recorded. Histidine decarboxylase exclusively was inhibited by N-termlnal histidine peptides. None of the other peptides investigated interfered negatively with this enzyme. This inhibition was consistent in the purified preparation and appeared to be more pronounced with increasing hydrophobieity in the second amino acid. Histidyl-phenylalanine was found to be about 100-fold as potent as the commonly used specific histldine deearboxylase inhibitor a-methyl histidine. It is concluded that small peptides with histidine as the N-termlnai amino acid might act as specific inhibitors for mammalian hlstidlne deearboxylase. An analog effect of small tyrosyl or phenylalanyl peptldes was not seen for the DOPA deearboxylase.
Introduction The biosynthesis of histamine proceeds via decarboxylation of histidine. The mammalian enzymes found to catalyse this reaction might differ in specificity. Histidine decarboxylase (EC 4.1.1.22) is characterized by a pH optimum at or below neutrality while an obviously different and less specific enzyme decarboxylates histidine at pH 8 or above [I-4, among others]. The latter enzyme might be identical with aromatic L-amino acid decarboxylase (EC 4.1.1.28) since its activity is blocked by the aromatic amino acid decarboxylase inhibitor orJ Presented in part at the 1 lth FEBS Meeting, Copenhagen, August 1977.
methyl DOPA [5, further refs. in 2]. Purified aromatic amino acid decarboxylase have been reported to, besides its preferred substrates DOPA and 5-hydroxy tryptophan, also to some extent act on histidine [6] but there are also contradictory results [4, 7, 8]. In attempts to find inhibitors specific for mammalian histidine decarboxylase (EC 4.1.1.22) different lines have been followed. Several imidazole and histidine analogs have been tried as potential histidine decarboxylase inhibitors [5, 9-16]. From these reports one might conclude that (i) substitution in the imidazole-ring is not allowed [9, I0], (it')/1-substitution is not allowed [I0, 1 I]. (iii) Substitution of a methyl group for the or-hydrogen yields an apparently specific inhibitor, or-methyl histidine (a, Scheme 1). An ethyl or higher alkyl group in the or-position gives compounds with less or no inhibitory effect on histidine decarboxylase [9, 11]. (iv) A free amino group is preferred, but might not be essential [9, 13], and (v) substitution at the carboxyl group in histidine has given some working inhibitors, among them the strongest reported among the histidine analogs; 4imidazolyl-3-amino-2-butanone [14, 15] (b, Scheme 1) and histidine methylester [9, 13] (c, Scheme 1). Apart from the histidine analogs, some benzoic acid derivatives [17, 18] and substituted phenols ]181, (+)Catechin [19[, some rhodanines [20] and methoxyamines [21-231 have been described as inhibitors for histidine decarboxylase. The methoxyamines, such as Brocresine (NSD 1055), are the strongest inhibitors known for the enzyme but are not specific for histidine decarboxylase. They are aldehydic reagents
315
Peptide Inhibition of Mammalian Histidine Decarboxylase
J7% ~ HIS
.."x
%,
17"'/0 I t \c.. (hi
~ (o1
HN--~~ 0 ["'N 1~)' O_--,CH3)3CH 3
To establish the specificity of their action comparison is made with their effects on aromatic L-amino acid decarboxylase, assayed as DOPA decarboxylase. The inhibitors a-methyl histidine and a-methyl DOPA of the respective decarboxylases, as well as several non-histidyldipeptides are included as controls.
Materials and methods
Enzymes
which apparently bind to the aldehyde group of the enzyme-bound pyridoxal phosphate, and therefore interferes with other pyridoxal phosphate-dependent enzymes as well. However, some selectivity has been observed with different methoxyamines. HUSZTI et al. [22] for instance, report that 3-nitrobenzyloxyamine and pyridyl-3methoxyamine have about 20-fold higher affinity for histidine decarboxylase than for aromatic Lamino acid decarboxylase, while some other benzyloxyamines and pyridylmethoxyamines were about equally potent with the two enzymes. The cited reports on histidine analog inhibition suggest that the substrate binding site is a narrow pocket highly specific for hlstidine. There is apparently some leeway in the catalytic site around the a-carbon, but only in the direction distal to histidine carbon-1 is there reasonable steric freedom as judged from the fact that alkylesters up to butyl are inhibitory [9]. The strong inhibition observed with benzyl and pyridyl substituted methoxyamines [22] suggests a hydrophobic area close to the catalytic center. Against this background it would seem likely that peptides with histidine at the amino terminus could act as inhibitors (d, Scheme 1). AURES and CLARK [24] found that histidyl-histidine did inhibit histidine decarboxylation in mouse mastocytoma to about the same extent as did ~methyl histidine but KELLEY et al. [9] tried histidylglycine as inhibitor to the rat stomach enzyme and found no effect at a peptide concentration of 0.1 raM. However, we have earlier noted a weak but apparently specific inhibition of histidine d e c a r b o x y l a s e b y histidyl-lysine a n d a histidylp e n t a p e p t i d e [25]. T h e p r e s e n t p a p e r c o n c e r n s the action o f several small histidyl-peptides o n the activity o f a m a m m a l i a n histidine d e c a r b o x y l a s e .
A crude extract of histidine and DOPA decarboxylase was prepared from a transplantable mast cell tumour of mice [26]; homogenization under argon was followed by centrifugation (150,000 x g, 2 h) and dialysis against 0.15 M potassium phosphate, 10 IJM pyridoxal phosphate, 1 mM dithiothreitol, 3 mM NaNj (pH 6.8). The specific activity of histidine decarboxylase in this crude extract was about 9 nmoles CO2/h.mg protein and the specific activity of DOPA decarboxylase was about 6 nmoles CO2/h.mg protein. Purification by hydroxyapatite chromatography, molecular sieving and chromatography on octyl Sepharose (to be published elsewhere) yielded a histidine decarboxylase with the specific activity of 1200 nmoles CO2/h.mg protein (the coefficient of variation in 8 different activity determinations was 9%). Only traces of DOPA decarboxylase activity was found in this partially purified preparation, which was used for the kinetic analysis of the effect of histidyl-phenylalanine on histidine decarboxylase.
Assay Decarboxylase activity was assayed as the amount ot 14CO2 released by the enzyme extract from (1-14C)-sub. strates at pH 6.8 and 37~ under argon during 1 h (DOPA decarboxylase) or 2 h (histidine decarboxylase) [261. The reaction mixture in a total volume of 50/J1 contained enzyme extract in amount to give an output in the histidinc decarboxylase controls (no inhibitor added) when assayed al 0.8 mM histidine of about 1300 cpm (1.2 nmoles CO2/h ) ir the case of the crude enzyme and about 2500 cpm (2.2 nmoles CO2/h) in the case of the purified enzyme (for th~ kinetic plot half this amount), L-histidine or L-DOPA, th( substance to be tested, 10 tJM pyridoxal phosphate, 1 m& dithiothreitol, 3 mM NAN3, 0.15 M potassium phosphat, (pH 6.8) and 40 nCi 1J4C-substrate. To minimize the erro: in the low-substrate activity measurement in the kineti, analyses the concentration of the substrate was varied whih the amount of radioactivity was kept constant. Thus th, blank level was kept within 30-50 cpm. The substrat~ concentration in the kinetic analysis was varied in the rang, 8-808 ~tM.
Chemicals The histidyl-dipeptides were from Bachem, Bubendor Schweiz. Their purity was confirmed by amino acid analys: after acid hydrolysis. The His. Arg. Ala. Ser. Val was syz thesized for other purposes and was available in the lab. L- i 14C-histidine and DL-I-~4C-DOPA were from New Englan Nuclear.
316
Peptide Inhibition of Mammalian Histidine Decarboxylase
Results
The effect of some small peptldes on the activity of histldine decarboxylase and DOPA decarboxylase Histidine decarboxylase and DOPA decarboxylase were assayed at saturating substrate concentrations (0.80 m M and 0.30 mM, respectively) in the presence of the compounds listed in Table 1. The results of the experiments with histidine decarboxylase are given in Table 1 and show that the histidine decarboxylase activity of both the crude extract and the purified enzyme is inhibited by histidyl-containing peptides. The non-histidylpeptides tested were not inhibitory. Among the Table 1
The effect of some small peptides, phenylalanine, a-methyl histidine and a-methyl D O P A on the activity of histidine decarboxylase in crude extract and in a partly purified preparation. Additive
Relative activity a as % of control with no additive Crude extract
compounds tested here the most effective inhibitor was the dipeptide His. Phe, closely followed by His. Tyr. Phenylalanine alone or in dipeptides lacking histidine had no effect. Since DOPA decarboxylase can under certain conditions decarboxylate histidine [6] it was of interest to determine whether this enzyme was also affected by the histidyl-dipeptides. In the presence of 0.5 m M c~-methyl DOPA the DOPA decarboxylase activity decreased to 42% of controls, but no significant effect was observed with any of the peptides listed or with 4 m M ~t-methyl histidine. Kinetics of the histidyl-phenylalanine inhibition of hlstldine decarboxylase The inhibition by histidyl-phenylalanine was further studied using the partially purified histidine decarboxylase. In a double reciprocal plot of kinetic data (Fig. 1) the lines intersect to the h,mg I nmol C02 /
Purified enzyme 31 ~
Concentration of additive: HIS. P H E HIS.TYR HIS. A L A HIS. HIS HIS.SER a-Methyl HIS HIS. LYS HIS. ASP HIS. A R G . A L A . SER. VAL LYS. VAL VAL. A L A GLY- LEU ALA. ALA ALA. GLY LYS.PHE PHE. VAL PHE. PHE e TRP. PHE e TYR. PHE c PHE a-Methyl D O P A
4 mM
0.2 m M
24
4 mM 3b 4b 23 b 25 b 39 b 40 b 42 b 79 b
25 22 31 40 27 32
0.8 m M
~ ....
I
/
100 I 200
12 b 19 b 52 b 64 b 75 b
~'00 ~ M / / /.DO
89 86 2O0
51 103 100 106 106 120 101 103
103
117
95
108 104 113 107 100
5
10
15
20
25 mM "1
1
100
a The average of four or more determinations are given. The standard error of the mean was below 5% of the given values except in the case of HIS. ALA, 0.8 mM, when it was 9%. b The lower limit of the normal distribution of the purified controls (n = 8) was 79% P < 0.01 and 73% P < 0.001. c Higher concentrations than 0.2 m M could not be tested due to the limited solubility of these peptides.
Figure 1 Double reciprocal kinetic plot of partially purified histidine decarboxylase at different concentrations of histidyl-phenyl-
alanine. Inserted: The slope and the 1IV intercepts of the lines from the double reciprocal plot vs. concentration of the peptide inhibitor. The lines are adopted by least square analysis to data obtained from triplicate determinations at five substrate concentrations (808, 178, 88, 38 and 8/zM, the last not shown in the figure) and the concentration of histidyl-phenylalanine in #molar as indicated on the lines.
317
Peptide Inhibition of Mammalian Histidine Decarboxylase
left of the 1/V axis, indicating that the inhibition might not be purely competitive. Secondary plots gives K~I~ = 44 # M and K~ntereept -- 244 #M. The apparent K m derived from the primary plot was 305 #M which is in agreement with what has been reported for the enzyme from the same and other sources when assayed under similar conditions [1-4, 27, 28]. All lines were derived by regression analysis. The regression coefficients are 0.98 and above, except in the case of the secondary plot of the 1/V intercepts where the regression coefficient was 0.96.
Discussion The concept that small peptides with the substrate amino acid as N-terminal might act as inhibitors for amino acid decarboxylases has not received much attention earlier. Our positive results for mammalian histldine decarboxylase might not hold for other enzymes. Two phenylalanyl, tryptophanyl and a tyrosyl dipeptide failed to inhibit the DOPA decarboxylase in our preparations. The inhibitory potency of the histidyldipeptides investigated apparently increases with increasing hydrophobicity in the second amino acid. This is consistent with the observation by MOLE et al. [18] that increasing hydrophobicity in m-substituted, benzoic acid derivatives correlates with increased inhibitory potency toward histidine decarboxylase. Comparison of our data with those given for the effect of histidine esters on rat stomach histidine decarboxylase [9] implies that the methylester is a stronger inhibitor than the histidyl-phenylalanine. The K I given for the histidine methylester was 1.8 #M, as compared to 44 #M found here for the histidyl-phenylalanine. However, the data might not be directly comparable since the enzyme sources differ and we needed almost a 10-fold higher concentration of the a-methyl histidine to get a relative inhibition of the mastocytomal enzyme comparable to that observed for the rat stomach enzyme. It is assumed that the histidine decarboxylation assayed is performed by the specific histidine decarboxylase (EC 4.1.1.22), since a-methyl DOPA had no effect on the observed histidine decarboxylation. Further evidence for this assumption is that the inhibition of histidine decarboxylation observed by a-methyl histidine and the histidyl-peptides is consistent in the purified preparation, where the DOPA de-
carboxylase activity is negligible compared to the histidine decarboxylase activity. The lack of effect of the histidyl-peptides on DOPA decarboxylase activity therefore strongly suggests that the inhibition observed is specific for the histidine decarboxylase. Small peptides in brain and other tissues have been shown to be regulatory. It is tempting to suggest that natural histidyl-peptides might operate on histidine decarboxylase in the regulation of histamine formation. Acknowledgments This work was supported by grants from the Finsen Foundation, Stockholm and Faculty of Science, Uppsala. The skilful technical assistance of Miss B. Ahlstrom is thankfully acknowledged. Received 15 January 1979
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Peptide Inhibition of Mammalian Histidine Decarboxylase
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