Mutation of an Active Site Residue of Tryptophan Synthase (β-Serine 377) Alters Cofactor Chemistry

Abstract

To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (␤-Ser 377 ) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pK a ϳ ϳ 7.7) from a form with a protonated internal aldimine nitrogen ( max ‫؍‬ 416 nm) to a deprotonated form ( max ‫؍‬ 336 nm), whereas the absorption spectra of the wild type tryptophan synthase ␤ 2 subunit and ␣ 2 ␤ 2 complex are pHindependent. The reaction of the S377D ␣ 2 ␤ 2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands ( max ‫؍‬ 498 -510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pK a of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.An important question in investigations of enzyme structure and function is how enzymes have evolved different reaction and substrate specificities. Pyridoxal phosphate (PLP) 1 -dependent enzymes are attractive targets for addressing this question because they catalyze a wide variety of reactions of amino acids (1, 2). The enzyme protein directs and restricts the catalytic potential of the bound PLP to provide the substrate and reaction specificity and the enhanced reaction rate of the enzyme (3, 4). Thus, it is important to understand how the protein structure controls the cofactor chemistry and enhances catalytic rates.Information on how the protein structure controls PLP-dependent reactions is beginning to emerge from x-ray crystallography and from sequence comparisons designed to establish evolutionary relationships (5, 6). One of the most important and best studied interactions between the cofactor and the protein active site of all PLP enzymes is that between the pyridine nitrogen of PLP and an amino acid side chain (see Fig.

Evidence for a Two-Base Mechanism Involving Tyrosine-265 from Arginine-219 Mutants of Alanine Racemase

“…Jhee et al (26) mutated the pyridine nitrogen-contacting serine in tryptophan synthetase (S377) to aspartate and found that a quinonoid intermediate accumulates. Grishin et al (8) proposed that ornithine decarboxylase has an R/ barrel fold like alanine racemase, yet this enzyme appears to have an aspartate residue interacting with the PLP pyridine nitrogen.…”

The Molecular Evolution of Pyridoxal‐5′‐Phosphate‐Dependent Enzymes

“…a positive charge at N1 is not a prerequisite for PLP to become catalytically effective. The effect of the group interacting with N1 on the electron distribution within the cofactor has been experimentally probed by site-directed mutagenesis experiments with aspartate aminotransferase of the a family (Yano et al, 1992;Onuffer and Kirsch, 1994) and the P-subunit of tryptophan synthase of the p family (Jhee et al, 1998). (3) K ions have been found to be essential for catalytic activity of particular B, enzymes such as 2,2-dialkylglycine decarboxylase (Toney et al, 1993(Toney et al, , 1995, tryptophan synthase P (Rhee et al, 1996), tyrosine phenol-lyase Sundararaju et al, 1997), and tryptophan phenol-lyase (tryptophanase; Isupov et al, 1998).…”

Reaction Intermediate Structures of 1-Aminocyclopropane-1-carboxylate Deaminase

“…The mutation of TRPS␤ introducing asparate or glutamate residues at the position of this serine residue stabilized the protonated form of pyridine nitrogen of PLP, reducing the pK a of the internal aldimine nitrogen and promoting the formation of quinonoid intermediate (40). Consequently, the mutant enzyme of TRPS␤ with asparate residue interacting with the pyridine nitrogen showed a pH-dependent absorption spectrum and mechanism-based inactivation by Lserine (40).…”

“…The mutation of TRPS␤ introducing asparate or glutamate residues at the position of this serine residue stabilized the protonated form of pyridine nitrogen of PLP, reducing the pK a of the internal aldimine nitrogen and promoting the formation of quinonoid intermediate (40). Consequently, the mutant enzyme of TRPS␤ with asparate residue interacting with the pyridine nitrogen showed a pH-dependent absorption spectrum and mechanism-based inactivation by Lserine (40). The absorption band shifted from 336 nm at around pH 9 to 416 nm at around pH 6.5, and incubation of the mutant enzyme with L-serine formed a covalent adduct of internal aldimine and aminoacrylate, which was confirmed by spectral data and a compound released from the adduct by alkaline.…”