The Crystal Structure of a hCA VII Variant Provides Insights into the Molecular Determinants Responsible for Its Catalytic Behavior.

Katia D’Ambrosio,Emma Langella,Simona Monti,Claudiu Supuran,Anna Di Fiore,Martina Buonanno,Giuseppina De Simone

doi:10.3390/ijms19061571

Katia D’Ambrosio, Emma Langella + Show 5 more

Open Access

https://doi.org/10.3390/ijms19061571

Copy DOI

Abstract

Although important progress has been achieved in understanding the catalytic mechanism of Carbonic Anhydrases, a detailed picture of all factors influencing the catalytic efficiency of the various human isoforms is still missing. In this paper we report a detailed structural study and theoretical pKa calculations on a hCA VII variant. The obtained data were compared with those already known for another thoroughly investigated cytosolic isoform, hCA II. Our structural studies show that in hCA VII the network of ordered water molecules, which connects the zinc bound solvent molecule to the proton shuttle His64, is altered compared to hCA II, causing a reduction of the catalytic efficiency. Theoretical calculations suggest that changes in solvent network are related to the difference in pKa of the proton shuttle in the two enzymes. The residue that plays a major role in determining the diverse pKa values of the proton shuttle is the one in position four, namely His for hCA II and Gly for hCA VII. This residue is located on the protein surface, outside of the active site cavity. These findings are in agreement with our previous studies that highlighted the importance of histidines on the protein surface of hCA II (among which His4) as crucial residues for the high catalytic efficiency of this isoform.

Highlights

Carbonic Anhydrases (CAs, EC 4.2.1.1) are ubiquitous metallo-enzymes that are present in most living organisms [1]
Abstract: important progress has been achieved in understanding the catalytic mechanism of Carbonic Anhydrases, a detailed picture of all factors influencing the catalytic efficiency of the various human isoforms is still missing
We recently focused our attention on Human CAs (hCAs) VII [30,31,32,33,34,35], an enzyme initially detected only in brain, where it functionally participates as a molecular switch for GABAergic excitation [30,36,37]

Summary

Introduction

Carbonic Anhydrases (CAs, EC 4.2.1.1) are ubiquitous metallo-enzymes that are present in most living organisms [1]. Human CAs (hCAs) belong to the α-class and exist in fifteen isoforms. Sharing a high level of three-dimensional similarity and a zinc ion in the active site, these isoforms differ in the tissue distribution, catalytic activity and cellular localization. Five isoforms are cytosolic (CAs I-III, VII and XIII), four are membrane-associated (CAs IV, IX, XII and XIV), two are mitochondrial (CAs VA and VB), and one is a secretory protein present in milk and saliva (CA VI) [1,9,10]. HCAs catalyze a simple but fundamental physiological reaction, the reversible hydration of CO2 to HCO3− and proton, following a two-step mechanism described by Equations (1) and (2) [1]. The binding of HCO3− to the zinc is rather labile, it is replaced by a water molecule

Methods

Results

Conclusion