Novel Catalytic Mechanism of Nucleophilic Substitution by Asparagine Residue Involving Cyanoalanine Intermediate Revealed by Mass Spectrometric Monitoring of an Enzyme Reaction

Abstract

. This enzyme has also been utilized as an industrial biocatalyst for the synthesis of chiral compounds based on its stereospecificity (2). We have studied intensely the reaction mechanism and structure of L-DEX YL, and revealed that the reaction begins with nucleophilic attack of Asp 10 on the ␣-carbon atom of L-2-haloalkanoates, causing the release of a halide ion and the formation of an ester intermediate (Scheme 1, I), which is subsequently hydrolyzed (3, 4). In the x-ray structure, there is a water molecule close to Asp 10 , Ser 175 , Asn 177 , and Asp 180 , the latter three of which are supposed to enhance the nucleophilicity of the water molecule for hydrolysis of the ester intermediate (5) (Scheme 1). These enzymological studies of L-DEX YL have provided critical information on the structures and functions of various hydrolases including P-type ATPases, which have a significant sequence similarity with L-DEX YL and are proposed to have the haloacid dehalogenase fold (6 -9). Recent studies on the crystal structure of Ca 2ϩ -ATPase from sarcoplasmic reticulum revealed that this ATPase has a high threedimensional structural similarity to that of L-DEX YL and many essential amino acid residues are conserved between these two proteins (10).In the course of the studies of L-DEX YL, we surprisingly found that replacement of the catalytic residue, Asp 10 , by Asn did not completely inactivate the enzyme, whereas replacement by Ala, Gly, Ser, or Glu resulted in total inactivation (11). One possible explanation is that the activity of the D10N preparation is attributed to the post-translational production of the wild-type enzyme by deamidation of Asn 10 : deamidation of Asn to produce Asp has been shown to occur in a variety of proteins (12, 13). However, it is also possible that the D10N enzyme itself catalyzes the hydrolysis of the substrate.In the present study, we analyzed the mechanism of dehalogenation catalyzed by the L-DEX YL D10N preparation by ion-spray mass spectrometry (MS), and found that the D10N enzyme itself, in addition to the wild-type enzyme produced by deamidation of the D10N enzyme, catalyzes dehalogenation. Asn 10 was shown to undergo a unique structural change in the course of the catalysis: it is converted into a ␤-cyanoalanine (␤-CNAla) residue via an imidate, and then regenerated in the catalytic cycle. This is the first report of the formation of a ␤-CNAla residue as an enzyme reaction intermediate. Also, the results demonstrate the validity of mass spectrometry in enzyme dynamics.

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

“…With the improvements of resolving power, sensitivity, and versatility, mass spectrometry has become a highly competitive analytical method for detection and characterization of biochemical reaction intermediates that can be used to elucidate enzymatic reaction pathways and catalytic mechanisms [16 -22]. Esaki et al reported a novel catalytic mechanism for L-2-Haloacid dehalogenase involving a cyanoalanine intermediate revealed by LC/MS monitoring of the enzymatic reaction [16]. Likewise, Leary and coworkers reported the identification of a sulfated NodH sulfotransferase (NodST) intermediate formed in a hybrid Ping-Pong mechanism using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry [17].…”

Involvement of Hat1p (Kat1p) Catalytic Activity and Subcellular Localization in Telomeric Silencing

“…Curiously mutation of Rtt109p Asp-287 and Asp-288 to alanine causes a significantly greater loss of catalytic activity than the corresponding asparagine mutants (45). As the asparagine residues are unlikely to function as catalytic bases, this may reflect the fact that post-translational conversion of asparagine to aspartic acid can occur in some proteins (55)(56)(57)(58).…”

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

“…YL (L-DEX YL) is one of the best studied enzymes of the HAD superfamily. 6,[9][10][11][12][13][14][15][16][17][18][19][20][21][22] Mass spectrometry has played an important role in analyzing the reaction mechanism of L-DEX YL.…”

“…2). 20,21) Upon incubation with the substrate L-2-chloropropionate, the peak corresponding to the native enzyme disappeared, and a new peak appeared at 26,255 Da (M þ 73), which is considered to be an ester intermediate. These results not only verified the validity of the proposed mechanism for L-DEX YL (Fig.…”