Roles for cationic residues at the quinolinic acid binding site of quinolinate phosphoribosyltransferase

Abstract

Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) forms nicotinate mononucleotide (NAMN) from quinolinic acid (QA) and 5-phosphoribosyl 1-pyrophosphate (PRPP). Previously determined crystal structures of QAPRTase.QA and QAPRTase.PA.PRPP complexes show positively charged residues (Arg118, Arg152, Arg175, Lys185, and His188) lining the QA binding site. To assess the roles of these residues in the Salmonella typhimurium QAPRTase reaction, they were individually mutated to alanine and the recombinant proteins overexpressed and purified from a recombineered Escherichia coli strain that lacks the QAPRTase gene. Gel filtration indicated that the mutations did not affect the dimeric aggregation state of the enzymes. Arg175 is critical for the QAPRTase reaction, and its mutation to alanine produced an inactive enzyme. The k(cat) values for R152A and K185A were reduced by 33-fold and 625-fold, and binding affinity of PRPP and QA to the enzymes decreased. R152A and K185A mutants displayed 116-fold and 83-fold increases in activity toward the normally inactive QA analogue, nicotinic acid (NA), indicating roles for these residues in defining the substrate specificity of QAPRTase. Moreover, K185A QAPRTase displayed a 300-fold higher k(cat)/K(m) for NA over the natural substrate QA. Pre-steady-state analysis of K185A with QA revealed a burst of nucleotide formation followed by a slower steady-state rate, unlike the linear kinetics of WT. Intriguingly, pre-steady-state analysis of K185A with NA produced a rapid but linear rate for NAMN formation. The result implies a critical role for Lys185 in the chemistry of the QAPRTase intermediate. Arg118 is an essential residue that reaches across the dimer interface. Mutation of Arg118 to alanine resulted in 5000-fold decrease in k(cat) value and a decrease in the binding affinity of QA and PRPP to R152A. Equimolar mixtures of R118A with inactive or virtually inactive mutants produced approximately 50% of the enzymatic activity of WT, establishing an interfacial role for Arg118 during catalysis.

Figure 1

Chemical structures of quinolinate and nicotinate.

Figure 2

Binding site for QA in the M. tuberculosis QAPRTase•PA•PRPCP•Mn²⁺ Michaelis complex (PDB entry 1QPR). Residues that interact with QA are shown and their hydrogen-bonding distances are represented in Å. The primes on Arg118′ designates residue from the adjacent subunit. C2 and C3 are designations of the homologous positions on QA. The Two Mn²⁺ are coordinated by PRPC are shown in gray. This figure was generated using SwissPDB Viewer (23).

Figure 3

Partial sequence alignment of QAPRTase from various organisms. 10 of the 230 sequences aligned as described in Methods are displayed. Sequences are: SALTY, Salmonella typhimurium LT2; ECOLI, Escherichia coli K-12; MYCTU, Mycobacterium tuberculosis H37Rv; HUMAN, Homo sapiens; SYNFM, Syntrophobacter fumaroxidans; MESSB, Mesorhizobium sp. BNC1; RHOPS, Rhodopseudomonas palustris BisB5; ERYLH, Erythrobacter litoralis; SYNS3, Synechococcus sp. CC9311; METST, Methanosphaera stadtmanae. Residue numbering at top is from the mature S. typhimurium protein sequence. Conserved residues are indicated in bold, absolutely conserved residues are marked with *, and highly conserved residues are marked with + underneath the alignments.

Figure 4

Scatchard plot for binding of R118A to [³H]QA. The value of K_D was 128 μM, with an n value of 1.9 mol of [³H]QA/mol QAPRTase dimer.

Figure 5

Pre-steady state kinetics of [³H]NAMN formation by R152A with [³H]QA. Rapid mixing was performed in a Kintek Chemical-Quench-Flow apparatus as described in Materials and Methods. The solid line represents a linear least square fit to the data points. The k_cat of the reaction was 0.1 s⁻¹.

Figure 6

[³H]NAMN formation by K185A with [³H]QA in the pre-steady state. Rapid mixing was performed manually as described in Materials and Methods. The curve represents the best fit of the data to equation 1, with rate constants equal to 0.01s⁻¹ and 0.00013 s⁻¹, for the pre-steady and steady states, respectively.

Figure 7

Pre-steady state kinetics of [¹⁴C]NAMN formation by K185A with [¹⁴C]NA. Rapid mixing was performed in the Kintek Chemical-Quench-Flow apparatus as described in Materials and Methods. The solid line represents a linear least square fit to the data points. The k_cat of the reaction was 0.03 s⁻¹.

Figure 8

Time dependent complementation of inactive R175A mutant. Poorly active R118A mutant was mixed with equimolar R175A as described in Materials and Methods. The line represents a fit of data to a single exponential function.