Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the ph optimum of a glycosidase 1 1Edited by M. F. Summers

Joshi, Manish D.; Sidhu, Gary; Pot, Isabelle; Brayer, G.D.; Withers, Stephen G.; McIntosh, Lawrence P.

doi:10.1006/jmbi.2000.3722

Cited by 216 publications

(158 citation statements)

References 55 publications

Supporting

Mentioning

141

Contrasting

Order By: Relevance

“…Furthermore, no protonation of Glu177 was found in N44D at pD 6.4 (Fig. 1B), which is even more surprising because NMR measurements indicated that the pK a of the general acid increases by about 1.5 pK units in the equivalent N35D variant of BCX relative to the WT enzyme (33,40).…”

Section: Discussionmentioning

confidence: 91%

See 1 more Smart Citation

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Wan

Parks

Hanson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pK a values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pK a values of the catalytic Glu residues in both the apo-and substrate-bound states of the enzyme. The calculated pK a of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.glycoside hydrolase | protonation state | macromolecular neutron crystallography | xylanase | molecular simulations

show abstract

Section: Discussionmentioning

confidence: 91%

“…It has been previously demonstrated that a single amino acid substitution of Asn35 to Asp in BCX reduces the pH optimum of activity from 5.7 to 4.6 (40). It was proposed that the N35D variant follows a reverse protonation mechanism (43).…”

Section: Discussionmentioning

confidence: 99%

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Wan

Parks

Hanson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…There are a few experimental examples of active site pK a values that have been changed (and thus the pH-activity profile re-engineered) to yield an efficient mutant enzyme (Thomas et al 1985;Meiering et al 1992;Loewenthal et al 1993;Cha and Batt 1998;Joshi et al 2000;Le Nours et al 2003;Hirata et al 2004;Kim et al 2006), but the pK a shifts have been modest and often the essential mutations have been found using comparative protein engineering strategies (i.e., mutations are introduced based on comparisons with a homologous enzyme that possesses the desired pH-activity profile). Therefore, the conclusion from two decade's work is that very specific point mutations in the active sites can change the pH dependence of enzymatic activity, but unless such specific active site point mutations are known (e.g., from comparative studies), there is not much hope of achieving a dramatic pH-activity profile shift with rational engineering methods.…”

mentioning

confidence: 99%

Redesigning protein pK_avalues

Tynan-Connolly

Nielsen

2007

Protein Science

View full text Add to dashboard Cite

The ability to re-engineer enzymatic pH-activity profiles is of importance for industrial applications of enzymes. We theoretically explore the feasibility of re-engineering enzymatic pH-activity profiles by changing active site pK a values using point mutations. We calculate the maximum achievable DpK a values for 141 target titratable groups in seven enzymes by introducing conservative net-charge altering point mutations. We examine the importance of the number of mutations introduced, their distance from the target titratable group, and the characteristics of the target group itself. The results show that multiple mutations at 10Å can change pK a values up to two units, but that the introduction of a requirement to keep other pK a values constant reduces the magnitude of the achievable DpK a . The algorithm presented shows a good correlation with existing experimental data and is available for download and via a web server at http://enzyme.ucd.ie/pKD.

show abstract

“…The movement of the inner loop ( 343 NFTCLEM 349 : corresponding to 343-349 in sweet potato) was reported to play critical roles in the catalytic mechanism and multiple-attack action of the enzyme in soybean (Kang et al 2005). Asn343 has important roles to the hydrogen-bond network, whose members are functionally linked to the catalytic residue and maintain active-site geometry in soybean (Fang and Ford 1998, Hirata et al 2004, Joshi et al 2000. From the study in soybean, Phe344, Thr345, and Cys346 are responsible for substrate binding and enzymatic activity control (Totsuka et al 1994, Pujadas andPalau 1997).…”

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence variation associated with .BETA.-amylase deficiency in the sweet potato Ipomoea batatas (L.) Lam

et al. 2009

View full text Add to dashboard Cite

Amyβ is a single gene that encodes β-amylase, a starch-hydrolyzing enzyme that confers sweetness to sweet potato roots upon cooking. To clarify the genetic basis underlying β-amylase null activity, we sequenced a highly conserved region of Amyβ, including the Amyβ active center, from normal and null cultivars. Comparison of the sequences revealed the presence of the In-Del sequences in null cultivars, which was thought to be a loss-of-function mutation causing a change from sweet to non-sweet. The In-Del frequency revealed using a multiple sequencing approach, and its association with β-amylase activity, showed that an inactive allele of Amyβ gene (Amyβ-I) exists. Amyβ expression analysis showed that Amyβ-I is transcribed normally, suggesting that translational control of β-amylase activity occurs in null mutants. The insertion in Amyβ-I caused a sequence frameshift resulting in an amino acid substitution with the incorporation of a stop codon in Amyβ-I. The Amyβ-I amino acid substitution and premature termination affect the substrate binding and catalytic site of the enzyme, which may result in an inactive protein that is responsible for the β-amylase inactivity in non-sweet cultivars. These findings are the basis of selection markers for non-sweetness in sweet potato breeding.

show abstract

Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the ph optimum of a glycosidase 1 1Edited by M. F. Summers

Cited by 216 publications

References 55 publications

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Redesigning protein pK_avalues

Nucleotide sequence variation associated with .BETA.-amylase deficiency in the sweet potato Ipomoea batatas (L.) Lam

Contact Info

Product

Resources

About

Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the ph optimum of a glycosidase 1 1Edited by M. F. Summers

Cited by 216 publications

References 55 publications

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Redesigning protein pKavalues

Nucleotide sequence variation associated with .BETA.-amylase deficiency in the sweet potato Ipomoea batatas (L.) Lam

Contact Info

Product

Resources

About

Redesigning protein pK_avalues