Aspartate transcarbamoylase molecules lacking one regulatory subunit

Area of Science:

• Biochemistry
• Enzymology
• Protein structure and function

Background:

• Aspartate transcarbamoylase (ATCase) is a key enzyme in pyrimidine biosynthesis.
• ATCase is a multimeric protein composed of catalytic and regulatory subunits.
• Understanding ATCase assembly and regulation is crucial for cellular metabolism.

Purpose of the Study:

• To investigate the reconstitution process of ATCase from isolated subunits.
• To characterize a stable intermediate component formed during reconstitution.
• To elucidate the role of regulatory subunits in ATCase structure and allosteric regulation.

Main Methods:

• Reconstitution of ATCase from isolated catalytic (c) and regulatory (r) subunits.
• Purification and molecular weight determination of the stable component (r(4)c(6)).
• Electrophoretic analysis of hybrid species formed by r(4)c(6) and regulatory subunits.
• Characterization of allosteric properties (sigmoidal kinetics, CTP inhibition).

Main Results:

• Formation of a stable component, r(4)c(6), during ATCase reconstitution with excess catalytic subunits.
• r(4)c(6) has a lower molecular weight than native ATCase and combines with regulatory subunits.
• r(4)c(6) contains two regulatory subunits crosslinking two catalytic trimers, differing from native enzyme's three regulatory subunits.
• The incomplete enzyme r(4)c(6) retains sigmoidal kinetics and CTP inhibition, but these effects are reduced.

Conclusions:

• The r(4)c(6) complex represents an intermediate in ATCase assembly/dissociation.
• This finding highlights the structural role of regulatory subunits in stabilizing ATCase.
• The study provides insights into the bonding domains governing ATCase stability and allosteric regulation.