Controlling active site loop dynamics in the (β/α)8 barrel enzyme indole-3-glycerol phosphate synthase

Kathleen F. O’Rourke, Aneta M. Jelowicki, David D. Boehr

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The β1α1 loop in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) is important for substrate binding, product release and chemical catalysis. IGPS catalyzes the ring closure of the substrate 1-(o-carboxyphenylamine)-1-dexoyribulose 5-phosphate to form indole-3-glycerol phosphate, involving distinct decarboxylation and dehydration steps. The ring closure step is rate-determining in the thermophilic Sulfolobus sulfataricus enzyme (ssIGPS) at high temperatures. The β1α1 loop is especially important in the dehydration step as it houses the general acid Lys53. We propose that loop dynamics are governed by competing interactions on the N-and C-terminal sides of the loop. We had previously shown that disrupting interactions with the N-terminal side of the loop through the N90A substitution decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics on the picosecond to millisecond timescales. Here, we show that disrupting interactions on the C-terminal side of the loop through the R64A/D65A substitutions likewise decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics. Interestingly, the triple substitution R64A/D65A/N90A leads to new µs–ms timescale loop dynamics and makes the ring-closure step rate-determining once again. These results are consistent with a model in which the β1α1 loop is maintained in a structurally dynamic state by these competing interactions, which is important for the dehydration step of catalysis. Competing interactions in other enzymes may likewise keep their loops and other structural elements appropriately mobile.

Original languageEnglish (US)
Article number129
JournalCatalysts
Volume6
Issue number9
DOIs
StatePublished - Sep 2016

Fingerprint

Indole-3-Glycerol-Phosphate Synthase
indoles
glycerols
Dehydration
Glycerol
enzymes
phosphates
Phosphates
Enzymes
Substitution reactions
dehydration
Catalysis
closures
disrupting
Substrates
Tryptophan
substitutes
catalysis
interactions
rings

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Physical and Theoretical Chemistry

Cite this

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title = "Controlling active site loop dynamics in the (β/α)8 barrel enzyme indole-3-glycerol phosphate synthase",
abstract = "The β1α1 loop in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) is important for substrate binding, product release and chemical catalysis. IGPS catalyzes the ring closure of the substrate 1-(o-carboxyphenylamine)-1-dexoyribulose 5-phosphate to form indole-3-glycerol phosphate, involving distinct decarboxylation and dehydration steps. The ring closure step is rate-determining in the thermophilic Sulfolobus sulfataricus enzyme (ssIGPS) at high temperatures. The β1α1 loop is especially important in the dehydration step as it houses the general acid Lys53. We propose that loop dynamics are governed by competing interactions on the N-and C-terminal sides of the loop. We had previously shown that disrupting interactions with the N-terminal side of the loop through the N90A substitution decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics on the picosecond to millisecond timescales. Here, we show that disrupting interactions on the C-terminal side of the loop through the R64A/D65A substitutions likewise decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics. Interestingly, the triple substitution R64A/D65A/N90A leads to new µs–ms timescale loop dynamics and makes the ring-closure step rate-determining once again. These results are consistent with a model in which the β1α1 loop is maintained in a structurally dynamic state by these competing interactions, which is important for the dehydration step of catalysis. Competing interactions in other enzymes may likewise keep their loops and other structural elements appropriately mobile.",
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Controlling active site loop dynamics in the (β/α)8 barrel enzyme indole-3-glycerol phosphate synthase. / O’Rourke, Kathleen F.; Jelowicki, Aneta M.; Boehr, David D.

In: Catalysts, Vol. 6, No. 9, 129, 09.2016.

Research output: Contribution to journalArticle

TY - JOUR

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AU - O’Rourke, Kathleen F.

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N2 - The β1α1 loop in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) is important for substrate binding, product release and chemical catalysis. IGPS catalyzes the ring closure of the substrate 1-(o-carboxyphenylamine)-1-dexoyribulose 5-phosphate to form indole-3-glycerol phosphate, involving distinct decarboxylation and dehydration steps. The ring closure step is rate-determining in the thermophilic Sulfolobus sulfataricus enzyme (ssIGPS) at high temperatures. The β1α1 loop is especially important in the dehydration step as it houses the general acid Lys53. We propose that loop dynamics are governed by competing interactions on the N-and C-terminal sides of the loop. We had previously shown that disrupting interactions with the N-terminal side of the loop through the N90A substitution decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics on the picosecond to millisecond timescales. Here, we show that disrupting interactions on the C-terminal side of the loop through the R64A/D65A substitutions likewise decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics. Interestingly, the triple substitution R64A/D65A/N90A leads to new µs–ms timescale loop dynamics and makes the ring-closure step rate-determining once again. These results are consistent with a model in which the β1α1 loop is maintained in a structurally dynamic state by these competing interactions, which is important for the dehydration step of catalysis. Competing interactions in other enzymes may likewise keep their loops and other structural elements appropriately mobile.

AB - The β1α1 loop in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) is important for substrate binding, product release and chemical catalysis. IGPS catalyzes the ring closure of the substrate 1-(o-carboxyphenylamine)-1-dexoyribulose 5-phosphate to form indole-3-glycerol phosphate, involving distinct decarboxylation and dehydration steps. The ring closure step is rate-determining in the thermophilic Sulfolobus sulfataricus enzyme (ssIGPS) at high temperatures. The β1α1 loop is especially important in the dehydration step as it houses the general acid Lys53. We propose that loop dynamics are governed by competing interactions on the N-and C-terminal sides of the loop. We had previously shown that disrupting interactions with the N-terminal side of the loop through the N90A substitution decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics on the picosecond to millisecond timescales. Here, we show that disrupting interactions on the C-terminal side of the loop through the R64A/D65A substitutions likewise decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics. Interestingly, the triple substitution R64A/D65A/N90A leads to new µs–ms timescale loop dynamics and makes the ring-closure step rate-determining once again. These results are consistent with a model in which the β1α1 loop is maintained in a structurally dynamic state by these competing interactions, which is important for the dehydration step of catalysis. Competing interactions in other enzymes may likewise keep their loops and other structural elements appropriately mobile.

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