Evidence of carbamoylphosphate induced conformational changes upon binding to human ornithine carbamoyltransferase

Abstract

SUMMARYHuman liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of omithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human omithine earbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subnnit.

Ornithine Transcarbamylase – From Structure to Metabolism: An Update

“…Subsequent ORN binding triggers an induced fit mechanism that finally moves the SMG loop to further shield the active site and fixes it in that position by direct interactions with ORN (Figure 2B). These conformational changes were evidenced already earlier by spectroscopic methods (Reichard, 1960;Colombo and Richterich, 1968;De Gregorio et al, 1999). The CP domains also provide all the subunit contacts that form the dish-like shape of OTC trimers (Figure 2A).…”

Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes

“…These results are consistent with earlier reports of crystal soaking experiments with E. coli OTCase in which CP cracked the unliganded crystals [36], and the difference ultraviolet absorption spectra of E. coli and yeast OTCases showing that CP binding alone produced the most significant spectral signal [17,37,38]. Recent ultraviolet spectroscopy experiments with human OTCase also demonstrate that binding of the first substrate, CP, induces a large protein isomerization [18].…”

“…This conformational change is similar to that which occurs in ATCase when N-phosphonacetyl--aspartate binds to the catalytic subunit of the enzyme [16]. However, differential ultraviolet absorption experiments show that binding of the first substrate, CP, to E. coli or human OTCase induces most of the conformational change [17,18], in contrast with ATCase where the ultraviolet absorbance change does not occur until the second substrate, -aspartate, binds [19]. In order to clarify the role of CP in inducing the conformational changes and fully understand substrate recognition by the enzyme, it was important to determine the structures of the binary complex of the enzyme with the first substrate bound.…”

Functional and structural characterization of ovine ornithine transcarbamoylaseElectronic supplementary information (ESI) available: a Ramachandran plot, and two figures illustrating statistics on the geometrical parameters of the structure. See http://www.rsc.org/suppdata/ob/b3/b304901a/

“…This behavior confirms the attainment of conformational change upon formation of enzyme-carbamoylphosphate or enzyme-inorganic phosphate binary complex. 14 When phosphate and -ornithine are both present the half-life of the enzyme increases (t 1/2 = 42 min), but the strongest protective effect is observed in the presence of carbamoylphosphate and -norvaline. This fact confirms that stabilizing changes of enzyme conformation take place during the formation of the carbamoylphosphate--ornithine-enzyme ternary complex.…”

“…The quaternary organisation of these enzymes is particularly relevant to its cooperative behaviour: kinetic and spectroscopic data suggest that the trimeric OTCase undergoes a conformational transition upon binding of carbamoyl phosphate, reaching a more stable three-dimensional structure. 13, 14 The human enzyme has been crystallised in two different states: in the presence of a bi-substrate analogue, in a conformation which is assumed to be an R state, and in the presence of only one of the two substrates, carbamoylphosphate, in a state that is possibly intermediate between the R and the T states. At present, the structure of an eukariotic trimeric OTCase in the T state is not yet available.…”