The structural basis of proteolytic activation of bovine glutamate dehydrogenase

Abstract

In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that came to light during those studies. We find that superactivation is caused by cleavage at the N terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in nontreated chymotrypsin may have led to the production of the larger 4-5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.Keywords: chymotrypsin; glutamate dehydrogenase; activation; guanosine triphosphate; adenosine triphosphateThe glutamate dehydrogenase of bovine liver, readily available commercially in a pure form from the 1950s onward, attracted the interest of enzymologists over many years because of its interesting and complex regulatory behavior (Olson and Anfinsen 1953;Talal and Tomkins 1964;Yielding et al. 1964;Engel and Dalziel 1969;Huang and Frieden 1969) cited by Monod et al. (1963) as one of the striking examples to establish the reality of allosteric control. Specifically, in addition to homotropic allosteric behavior with the oxidized and reduced coenzymes (Olson and Anfinsen 1953;Yielding et al. 1964;Huang and Frieden 1969), the enzyme also exhibited potent inhibition by GTP and activation by ADP (Wolff 1962;Hershko and Kindler 1966). This led to much experimentation and controversy over the number and location of nucleotide sites and the relationship between them (Markau et al. 1971;Koberstein and Sund 1973;Bayley and O'Neill 1980). Chemical modification experiments (Colman and Frieden 1966;Price and Radda 1969;Coffee et al. 1971) uniformly showed that the response to purine nucleotides was associated with the C-terminal 50 amino acids of the protein, a finding that correlated well with the finding that homologous GDHs of prokaryotes and fungi lacked both the 50-amino acid section and the regulation by GTP/ADP (Wootton et al. 1974;Mattaj et al. 1982). Latterly, the recent solution of the threedimensional structure of bovine GDH (Peterson and Smith 1999) has revealed that these 50 amino acids form an ''antenna'' protruding from the main body of the hexameric structure and totally missing in various other solved GDH structures (Rice et al. 1987;Stillman et al. 1995

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes

“…This hypothesis is supported by the structural features of GDH as shown in Fig. 4b ; Asp6 and Asn8 form direct contacts with Lys329 through H-bonds and salt bridges, which therefore prevents the separation of fragments from the N-terminal region 36 . In addition, limited digestion experiments by Banerjee et al .…”

A novel mechanism of V‐type zinc inhibition of glutamate dehydrogenase results from disruption of subunit interactions necessary for efficient catalysis

“…In addition to the data presented above, previous MALDI studies demonstrated that the motility of this loop is diminished when the enzyme is locked into an abortive complex [30,31] and the α‐helix immediately upstream from this loop moves as the catalytic cleft opens and closes [30,31]. Recent studies have shown that chymotrypsin cleavage in this region removes this helical region, resulting in an activated form of the enzyme [41]. Finally, we recently determined the structures of two different drug–GDH complexes; these potent inhibitors were found to bind in the immediate vicinity of this zinc binding site.…”

Bovine glutamate dehydrogenase immobilization on magnetic nanoparticles: conformational changes and catalysis

“…19,20 As a consequence, any modication of these mechanisms through immobilization can alter the precise distribution of states within the native state ensemble 21,22 and modulate the allosteric response of the whole system. An example of a useful multimeric enzyme is the 336 kDa homohexameric protein glutamate dehydrogenase (GDH) from bovine liver, 23,24 which has its activity tightly controlled by a complex network of allosteric regulators 23,25 and its immobilization can induce differences in allosteric response. 26 In its immobilized form, glutamate dehydrogenase is normally used as glutamate probes, [27][28][29][30] employing different supports and methodologies for the attachment on a support.…”