Electrostatics of mesophilic and psychrophilic trypsin isoenzymes: Qualitative evaluation of electrostatic differences at the substrate binding site

Abstract

A qualitative evaluation of electrostatic features of the substrate binding region of seven isoenzymes of trypsin has been performed by using the continuum electrostatic model for the solution of the Poisson-Boltzmann equation. The sources of the electrostatic differences among the trypsins have been sought by comparative calculations on selective charges: all charges, conserved charges, partial charges, unique cold trypsin charges, and a number of charge mutations. As expected, most of the negative potential at the S(1) region of all trypsins is generated from Asp(189), but the potential varies significantly among the seven trypsin isoenzymes. The three cold active enzymes included in this study possess a notably lower potential at and around the S(1)-pocket compared with the warm active counterparts; this finding may be the main contribution to the increased binding affinity. The source of the differences are nonconserved charged residues outside the specificity pocket, producing electric fields at the S(1)-pocket that are different in both sign and magnitude. The surface charges of the mesophilic trypsins generally induce the S(1) pocket positively, whereas surface charges of the cold trypsins produce a negative electric field of this region. Calculations on mutants, where charged amino acids were substituted between the trypsins, showed that mutations in Loop2 (residues 221B and 224) and residue 175, in particular, were responsible for the low potential of the cold enzymes.

Psychrophilic Enzymes: From Folding to Function and Biotechnology

“…Electrostatic surface potentials generated by charged and polar groups are an essential component of the catalytic mechanism at various stages: as the potential extends out into the medium, a substrate can be oriented and attracted before any contact between enzyme and substrate occurs. Interestingly, the cold-active citrate synthase [124], malate dehydrogenase [131], uracil-DNA glycosylase [151, 172, 173], elastase [174], and trypsin [175–177] are characterized by marked differences in electrostatic potentials near the active site region compared to their mesophilic or thermophilic counterparts that may facilitate interaction with ligand. In the case of malate dehydrogenase, for example, the increased positive potential at and around the oxaloacetate binding site and the significantly decreased negative surface potential at the NADH binding region may facilitate the interaction of the oppositely charged ligands with the surface of the enzyme [131].…”

Protein stability and enzyme activity at extreme biological temperatures

“…Electrostatic surface potentials generated by charged and polar groups are an essential component of the catalytic mechanism at various stages: as the potential extends out into the medium, a substrate can be oriented and attracted before any contact between enzyme and substrate occurs. Interestingly, the cold-active citrate synthase [43], malate dehydrogenase [59], uracil-DNA glycosylase [60] and trypsin [35,61,62] are characterized by marked differences in electrostatic potentials near the active site region compared to their mesophilic or thermophilic counterparts that may facilitate interaction with oppositely charged ligands. In all cases, the differences were caused by discrete substitutions in non-conserved charged residues resulting in local electrostatic potential differing in both sign and magnitude.…”

Psychrophilic enzymes: strategies for cold-adaptation

“…Thus, substrates can be accommodated and products released at a lower energy cost due to the reduced conformational changes required, but, on the other hand, looser substrate binding may also occur leading to a higher K m . In contrast with this, optimization of the electrostatic potential of the active site [30,31,34,68,69] can allow for improved substrate recognition and binding and a reduction in K m . Here, the electrostatic potential of the active site can extend out into the medium and attract and orient the substrate even before this enters in contact with the enzyme while also enhancing binding.…”