Glutamic Aspartic Transaminase

Jenkins, W. Terry; Sizer, Irwin W.

doi:10.1021/ja01567a086

Cited by 129 publications

(47 citation statements)

References 49 publications

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“…18). The binding of maleate to WT causes an increase in the 430-nm absorption band with a concomitant decrease in the 360-nm band, reflecting the increase in the aldimine pK a value (17). Similar spectral changes are observed for the binding of maleate to V39F (data not shown).…”

Section: Resultssupporting

confidence: 69%

“…Aldimine pK a Values-The absorption spectra of the V39F mutant AspAT (V39F) show an absorption maximum at 358 nm in the alkaline pH region and at 430 nm in the acidic pH region (data not shown), which are identical to those of the WT AspAT and correspond to the unprotonated (E L ; see Scheme I) and protonated (E L H ϩ ; see Scheme I) structures of the PLPLys 258 aldimine, respectively (13,17). The pH dependence of the apparent molar absorptivity at 430 nm is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The intrinsic pK a of the aldimine has been estimated to be 8.8, by spectroscopic analysis of the maleate-bound enzyme, and it has been explained that the increase in the pK a is because of the electrostatic effect of the anionic ligand (17,18). However, taking into account the relatively weak electrostatic effect (0.7 pH unit) of Arg 292 * and Arg 386 on the aldimine pK a value as described above, we must consider other mechanisms for the upward pK a shift in this complex, such as alterations in the torsion angle of the aldimine and the hydrogen bond network surrounding it.…”

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confidence: 99%

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Conformational Change in Aspartate Aminotransferase on Substrate Binding Induces Strain in the Catalytic Group and Enhances Catalysis

Hayashi¹,

Mizuguchi²,

Miyahara³

et al. 2003

Journal of Biological Chemistry

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Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK a of the pyridoxal 5-phosphate-Lys 258 aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK a and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val 39 located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK a was lowered by 0.7 unit compared with the wildtype enzyme. When Asn 194 had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK a , showing that the domain rotation controls the aldimine pK a via the Arg 386 -Asn 194 -pyridoxal 5-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK a 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn 194 , and Arg 386 were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK a value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly The homodimeric structures have been solved for cytosolic (2), mitochondrial (3-5), and Escherichia coli (6, 7) enzymes. Each subunit is composed of large and small domains, and the active site containing the PLP-Lys 258 aldimine is located between the two domains. The ␣-carboxylate group of the dicarboxylic substrates binds to Arg 386 located at the small domain and the -carboxylate group to Arg 292 * located at the large domain. An asterisk indicates that the residue comes from the neighboring subunit. On the basis of structural, steady-state (1), and transient (8, 9) kinetic studies, the reaction mechanism of AspAT has been proposed (10, 11) and refined (9) (see Scheme I).The PLP-Lys 258 aldimine (internal aldimine) of the unliganded AspAT has an imine pK a value of ϳ6.5, which is strikingly lower than those of the PLP-amine aldimines with protonated pyridine N (ϳ10; see Ref 13). We have shown that the principle factor that decreases the aldimine pK a value of AspAT is the imine-pyridine torsion of the PLP-Lys 258 aldimine (as represented by the torsion angle C3-C4 -C4Ј-N, expressed as , in the panel of Scheme I), rather than the historically accepted electrostatic effect of the neighboring positive charges of Arg 292 * and Arg 386 . The torsion of the aldimine, the angle of which spans the range from ϳ35°(protonated aldimine) (2) to ϳ90°(unprotonated aldimine) (7), causes a decrease of 3 units in the aldimine pK a (14), whereas the positive charges of the arginine residues decreases the...

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Section: Resultssupporting

confidence: 69%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Conformational Change in Aspartate Aminotransferase on Substrate Binding Induces Strain in the Catalytic Group and Enhances Catalysis

Hayashi¹,

Mizuguchi²,

Miyahara³

et al. 2003

Journal of Biological Chemistry

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“…1B). AspAT has absorption bands in the region 300 -500 nm, which derive from the bound PLP molecule and are influenced significantly by amino acid residues interacting with PLP (11,26,27). The wild-type AspAT and AV5A-7 exhibit two major bands around 360 and 430 nm, whereas in ATB17 the latter band is red-shifted to 450 nm and has a broad shoulder above 500 nm.…”

Section: Resultsmentioning

confidence: 99%

Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues

Oue

Okamoto

Yano

et al. 1999

Journal of Biological Chemistry

178

100

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Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 ؋ 10 6 -fold increase in the catalytic efficiency (k cat /K m ) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-Å resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.

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“…The depressing effect was pH dependent, being greater at higher pH's. The Schiff's base formation is also known to be stabler at higher pH's (MORINO and SNELL, 1967;JENKINS and SIZER, 1957). These findings again suggested that the lysine residues are important in controlling the opening of C1-channels at this receptor complex.…”

Section: ) P-nitrothiophenol (1 Mm)mentioning

confidence: 85%

Effects of various enzymes and chemical modification reagents on the Na+- and Cl--dependent responses of the ganglion cells to acetylcholine.

et al. 1983

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Using the abdominal ganglion cells of Aplysia, we analyzed the effects of various enzymes and chemical modification reagents on the acetylcholine (ACh)-induced responses of the excitatory (Natdependent) and inhibitory (Cl--dependent) types. (1) Phospholipase A (2 mg/ml) caused no appreciable effects on either type of response.(2) Phospholipase C (2 mg/ml) markedly depressed both types of response. These suggested that the phosphoryl group of the phospholipid is an important site related to the binding of ACh, common to both types of ACh-receptors. (3) Carboxypeptidase A (10 mg/ml) caused no observable effects on either type of response. (4) Carboxypeptidase B (10 mg/ml) depressed the inhibitory type of response without affecting the excitatory one. (5) Pyridoxal-5'-phosphate (1 mM) also depressed the inhibitory response without affecting the excitatory one. These findings (4, 5) suggested that the Cl--channel in the inhibitory AChreceptor complex includes a C-terminal lysine which may play an active role in the movement of Cl-across the receptor membrane. (6) LLeucine aminopeptidase (1 mg/ml) depressed the excitatory response without altering the inhibitory one. (7) p-Nitrothiophenol (1 mM) also depressed the excitatory response without affecting the inhibitory one. These findings (6, 7) suggested the presence of a certain N-terminal amino acid near a glutamate or aspartate residue within a molecular moiety of Na+-channel included in the excitatory ACh-receptor complex.

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Glutamic Aspartic Transaminase

Cited by 129 publications

References 49 publications

Conformational Change in Aspartate Aminotransferase on Substrate Binding Induces Strain in the Catalytic Group and Enhances Catalysis

Conformational Change in Aspartate Aminotransferase on Substrate Binding Induces Strain in the Catalytic Group and Enhances Catalysis

Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues

Effects of various enzymes and chemical modification reagents on the Na+- and Cl--dependent responses of the ganglion cells to acetylcholine.

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