Electrostatic Preorganization in Three Distinct Heterogeneous Proteasome β‑Subunits

The origin of the
enzyme’s powerful role in accelerating
chemical reactions is one of the most critical and still widely discussed
questions. It is already accepted that enzymes impose an electrostatic
field onto their substrates by adopting complex three-dimensional
structures; therefore, the preorganization of electric fields inside
protein active sites has been proposed as a crucial contributor to
catalytic mechanisms and rate constant enhancement. In this work,
we focus on three catalytically active β-subunits of 20S proteasomes
with low sequence identity (∼30%) whose active sites, although
situated in an electrostatically miscellaneous environment, catalyze
the same chemical reaction with similar catalytic efficiency. Our
in silico experiments reproduce the experimentally observed equivalent
reactivity of the three sites and show that obliteration of the electrostatic
potential in all active sites would deprive the enzymes of their catalytic
power by slowing down the chemical process by a factor of 1035. To regain enzymatic efficiency, besides catalytic Thr1 and Lys33
residues, the presence of aspartic acid in position 17 and an aqueous
solvent is required, proving that the electrostatic potential generated
by the remaining residues is insignificant for catalysis. Moreover,
it was found that the gradual decay of atomic charges on Asp17 strongly
correlates with the enzyme’s catalytic rate deterioration as
well as with a change in the charge distributions due to introduced
mutations. The computational procedure used and described here may
help identify key residues for catalysis in other biomolecular systems
and consequently may contribute to the process of designing enzyme-like
synthetic catalysts.

酶在加速化学反应过程中发挥强效作用的根源，是学界最核心且仍被广泛探讨的问题之一。目前学界已达成共识，酶通过形成复杂的三维结构，会对其底物施加静电场；因此，蛋白质活性位点内部电场的预组织效应，被认为是催化机制与速率常数提升的关键促成因素。本研究聚焦于三个序列同一性约为30%的20S蛋白酶体（20S proteasomes）催化活性β亚基（β-subunits），尽管它们的活性位点处于静电异质性环境中，却能以相近的催化效率催化同一化学反应。我们的计算机模拟实验（in silico experiments）复现了实验观测到的三个活性位点的等效反应性，并证实：若消除所有活性位点内的静电势，化学反应速率将降低10³⁵倍，进而剥夺酶的催化能力。为恢复酶的催化效率，除催化相关的苏氨酸1（Thr1）与赖氨酸33（Lys33）残基外，还需要17号位的天冬氨酸残基以及水溶液环境，这证明其余残基产生的静电势对催化作用无显著贡献。此外，研究发现17号位天冬氨酸残基上的原子电荷逐渐衰减，与酶催化速率的下降以及引入突变后电荷分布的变化均呈现强相关性。本文所采用并阐述的计算流程，或可助力识别其他生物分子系统中的催化关键残基，进而推动类酶合成催化剂的设计进程。