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. 1974 Aug;71(8):3221–3225. doi: 10.1073/pnas.71.8.3221

Pathways of Assembly of Aspartate Transcarbamoylase from Catalytic and Regulatory Subunits

M Bothwell 1,*, H K Schachman 1,†
PMCID: PMC388655  PMID: 4606892

Abstract

A scheme for the assembly of aspartate transcarbamoylase (EC 2.1.3.2; carbamoylphosphate:L-aspartate carbamoyltransferase) from catalytic and regulatory subunits is presented along with a technique for detecting intermediates and measuring the kinetics of assembly at protein concentrations of about 10 nM. 125I-labeled subunits (106 cpm/μg) were used, and the reaction was “topped”at specific times with an appropriate “chase” followed by electrophoretic separation and measurement of the amounts of the various species. Two intermediates were identified. A stable enzyme complex lacking one regulatory subunit is the principal product when catalytic subunits are in excess. A transiently stable complex lacking one catalytic subunit is the principal species when regulatory subunits are in excess. Measurements with mixtures of the purified regulatory-deficient molecules and free regulatory subunits gave a second-order rate constant of 105 105 M-1 sec-1 for the formation of bonding domains between catalytic and regulatory chains. No data for the rate of rupture of these bonds are available, but the kinetics of the assembly of the enzyme in vitro can be accounted for if this value is about 10-2 sec-1. Assembly from subunits occurs in seconds at concentrations equivalent to those that would exist in vitro even if there were only enzyme molecule per cell.

Keywords: protein interactions, bonding domains, allosteric enzymes, kinetics

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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