New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate

Orellana, María-Soledad; Jaña, Gonzalo A.; Figueroa, Maximiliano; Martínez-Oyanedel, José; Medina, Fabiola E.; Tarifeño-Saldivia, Estefanía; Gatica, Marcell; García-Robles, María Ángeles; Carvajal, Nelson; Uribe, Elena

doi:10.3390/ijms23126438

Open AccessArticle

New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate

by

María-Soledad Orellana

^1,2,

Gonzalo A. Jaña

³

,

Maximiliano Figueroa

¹

,

José Martínez-Oyanedel

¹

,

Fabiola E. Medina

⁴

,

Estefanía Tarifeño-Saldivia

¹,

Marcell Gatica

¹,

María Ángeles García-Robles

⁵,

Nelson Carvajal

¹ and

Elena Uribe

^1,*

¹

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4070386, Chile

²

Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago 8370251, Chile

³

Departamento de Ciencias Químicas, Facultad Ciencias Exactas, Universidad Andres Bello, Autopista Concepción-Talcahuano 7100, Concepción 4070386, Chile

⁴

Departamento de Química, Facultad de Ciencias, Universidad del Bio-Bio, Concepción 4051381, Chile

⁵

Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción 4070386, Chile

^*

Author to whom correspondence should be addressed.

Int. J. Mol. Sci. 2022, 23(12), 6438; https://doi.org/10.3390/ijms23126438

Submission received: 28 April 2022 / Revised: 25 May 2022 / Accepted: 4 June 2022 / Published: 9 June 2022

(This article belongs to the Special Issue Ligand Binding in Enzyme Systems 2.0)

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Abstract

Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea. This enzyme has several analogies with agmatinase, which catalyzes the hydrolysis of agmatine into putrescine and urea. However, this contrasts with the highlighted specificity that each one presents for their respective substrate. A comparison of available crystal structures for arginases reveals an important difference in the extension of two loops located in the entrance of the active site. The first, denominated loop A (I129-L140) contains the residues that interact with the alpha carboxyl group or arginine of arginase, and the loop B (D181-P184) contains the residues that interact with the alpha amino group of arginine. In this work, to determine the importance of these loops in the specificity of arginase, single, double, and triple arginase mutants in these loops were constructed, as well as chimeras between type I human arginase and E. coli agmatinase. In previous studies, the substitution of N130D in arginase (in loop A) generated a species capable of hydrolyzing arginine and agmatine. Now, the specificity of arginase is completely altered, generating a chimeric species that is only active with agmatine as a substrate, by substituting I129T, N130Y, and T131A together with the elimination of residues P132, L133, and T134. In addition, Quantum Mechanic/Molecular Mechanic (QM/MM) calculations were carried out to study the accommodation of the substrates in in the active site of this chimera. With these results it is concluded that this loop is decisive to discriminate the type of substrate susceptible to be hydrolyzed by arginase. Evidence was also obtained to define the loop B as a structural determinant for substrate affinity. Concretely, the double mutation D181T and V182E generate an enzyme with an essentially unaltered k_cat value, but with a significantly increased K_m value for arginine and a significant decrease in affinity for its product ornithine.

Keywords: arginase; arginine; agmatine; determinants of specificity

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MDPI and ACS Style

Orellana, M.-S.; Jaña, G.A.; Figueroa, M.; Martínez-Oyanedel, J.; Medina, F.E.; Tarifeño-Saldivia, E.; Gatica, M.; García-Robles, M.Á.; Carvajal, N.; Uribe, E. New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate. Int. J. Mol. Sci. 2022, 23, 6438. https://doi.org/10.3390/ijms23126438

AMA Style

Orellana M-S, Jaña GA, Figueroa M, Martínez-Oyanedel J, Medina FE, Tarifeño-Saldivia E, Gatica M, García-Robles MÁ, Carvajal N, Uribe E. New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate. International Journal of Molecular Sciences. 2022; 23(12):6438. https://doi.org/10.3390/ijms23126438

Chicago/Turabian Style

Orellana, María-Soledad, Gonzalo A. Jaña, Maximiliano Figueroa, José Martínez-Oyanedel, Fabiola E. Medina, Estefanía Tarifeño-Saldivia, Marcell Gatica, María Ángeles García-Robles, Nelson Carvajal, and Elena Uribe. 2022. "New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate" International Journal of Molecular Sciences 23, no. 12: 6438. https://doi.org/10.3390/ijms23126438

APA Style

Orellana, M.-S., Jaña, G. A., Figueroa, M., Martínez-Oyanedel, J., Medina, F. E., Tarifeño-Saldivia, E., Gatica, M., García-Robles, M. Á., Carvajal, N., & Uribe, E. (2022). New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate. International Journal of Molecular Sciences, 23(12), 6438. https://doi.org/10.3390/ijms23126438

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New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate

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