pH studies toward the elucidation of the auxiliary catalyst for pig heart aspartate aminotransferase

Kiick, Dennis M.; Cook, Paul F.

doi:10.1021/bi00271a022

Cited by 87 publications

(83 citation statements)

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“…In humans, there are two isozymes, a mitochondrial (hBCATm) and a cytosolic (hBCATc) form (1-6), whereas bacteria contain only a single BCAT (6). The BCAT and transamination reactions of other pyridoxal 5Ј-phosphate (PLP)-dependent enzymes follow a ping-pong Bi-Bi reaction (7,8).Amino acid 1 ϩ ␣-keto acid 2^␣ -keto acid 1 ϩ amino acid 2 …”

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“…The reaction is accompanied by interconversion of the cofactor between the PLP and the pyridoxamine 5Ј-phosphate (PMP) forms (7)(8)(9)(10)(11)(12). In the first half-reaction, the PLP form of BCAT reacts with the branched chain amino acid, and the reaction proceeds through a Michaelis complex, an external aldimine, quinonoid intermediate, ketimine, and finally the PMP form of the BCAT and the branched chain ␣-keto acid product.…”

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Human Mitochondrial Branched Chain Aminotransferase Isozyme

Yennawar

Islam

Conway

et al. 2006

Journal of Biological Chemistry

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Mammalian branched chain aminotransferases (BCATs) have a unique CXXC center. Kinetic and structural studies of three CXXC center mutants (C315A, C318A, and C315A/C318A) of human mitochondrial (hBCATm) isozyme and the oxidized hBCATm enzyme (hBCATm-Ox) have been used to elucidate the role of this center in hBCATm catalysis. X-ray crystallography revealed that the CXXC motif, through its network of hydrogen bonds, plays a crucial role in orienting the substrate optimally for catalysis. In all structures, there were changes in the structure of the ␤-turn preceding the CXXC motif when compared with wild type protein.The N-terminal loop between residues 15 and 32 is flexible in the oxidized and mutant enzymes, the disorder greater in the oxidized protein. Disordering of the N-terminal loop disrupts the integrity of the side chain binding pocket, particularly for the branched chain side chain, less so for the dicarboxylate substrate side chain. The kinetic studies of the mutant and oxidized enzymes support the structural analysis. The kinetic results showed that the predominant effect of oxidation was on the second half-reaction rather than the first half-reaction. The oxidized enzyme was completely inactive, whereas the mutants showed limited activity. Model building of the second half-reaction substrate ␣-ketoisocaproate in the pyridoxamine 5-phosphate-hBCATm structure suggests that disruption of the CXXC center results in altered substrate orientation and deprotonation of the amino group of pyridoxamine 5-phosphate, which inhibits catalysis. Branched chain aminotransferases (BCATs)4 (EC 2.6.1.42) catalyze transamination of the branched chain amino acids, leucine, isoleucine, and valine, to their respective ␣-keto acids, ␣-ketoisocaproate, ␣-keto-␤-methylvalerate, and ␣-ketoisovalerate. In humans, there are two isozymes, a mitochondrial (hBCATm) and a cytosolic (hBCATc) form (1-6), whereas bacteria contain only a single BCAT (6). The BCAT and transamination reactions of other pyridoxal 5Ј-phosphate (PLP)-dependent enzymes follow a ping-pong Bi-Bi reaction (7,8).Amino acid 1 ϩ ␣-keto acid 2^␣ -keto acid 1 ϩ amino acid 2 REACTION 1The reaction is accompanied by interconversion of the cofactor between the PLP and the pyridoxamine 5Ј-phosphate (PMP) forms (7-12). In the first half-reaction, the PLP form of BCAT reacts with the branched chain amino acid, and the reaction proceeds through a Michaelis complex, an external aldimine, quinonoid intermediate, ketimine, and finally the PMP form of the BCAT and the branched chain ␣-keto acid product. In the Michaelis complex, the ␣-amino group of the substrate must be deprotonated to form a new Schiff base with PLP. The migration of the proton on the ␣-amino group of the substrate to the short contact pair between the ␣-carboxylate and the phosphate of PLP reduces the electrostatic repulsion between the pair and renders the ␣-amino group of the substrate deprotonated (6). The second half-reaction is the reverse of the first half-reaction.Structurally PLP-dependent enzymes are c...

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Human Mitochondrial Branched Chain Aminotransferase Isozyme

Yennawar

Islam

Conway

et al. 2006

Journal of Biological Chemistry

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“…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 pK a by only 0.7 unit (15). The catalytic significance of the aldimine torsion is considered to be that it increases the energy level of the protonated form of the aldimine in the unliganded enzyme, thereby decreasing the 1 The abbreviations used are: AspAT, aspartate aminotransferase (aspartate, 2-oxoglutarate aminotransferase, EC 2.6.1.1); PLP, pyridoxal 5Ј-phosphate; PMP, pyridoxamine 5Ј-phosphate; V39F AspAT, mutant AspAT in which the residue Val 39 has been replaced with a phenylalanine residue (other mutant AspATs are expressed in the same way); WT, wild-type; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid.…”

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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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“…Aminotransferases are well known pyridoxal 5Ј-phosphate (PLP 1 )-dependent enzymes that catalyze the reversible transfer of the amino group from an amino acid to a 2-keto acid (1)(2)(3)(4)(5),…”

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