Phani Kumar Pullela

Structural evidence for a functionally relevant second camphor binding site in P450cam: Model for substrate entry into a P450 active site

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F‐G helix that covers the active site pocket. Such a two‐step binding process is kinetically required if P450cam rarely populates an open conformation—as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor‐bound P450cam. The docking identified only one potential entry site pocket, a well‐defined cavity on the F‐helix side of the F‐G flap, 16 Å from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation‐based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [1H‐13C] HSQC titrations of 13CH3‐threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, 13CH3S‐pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam‐pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 μM. The two‐site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered‐substrates, which force a partially opened conformation. Proteins 2007.

A Dithio-Coupled Kinase and ATPase Assay

Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPβS (sulfur on the β-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPβS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPβS and not with ATPβS. Thiol detection was done using both Ellman’s reagent (DTNB) and a recently developed fluorescent dithio reagent, DSSA. Therefore, the assay can be run in both absorbance and fluorescence detection modes. The assay was used to perform steady-state kinetic analyses of both hexokinase and myosin ATPase. It was also used to demonstrate the diastereoselectivity of hexokinase (R) and myosin ATPase (S) for the isomers of ATPβS, consistent with previous results. When run in fluorescence mode using a plate reader, an average Z′ value of 0.54 was obtained, suggesting the assay is appropriate for high-throughput screening.

13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a 13CH3-reporter attached. This 13C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site.

The synthesis, characterization, and application of 13C-methyl isocyanide as an NMR probe of heme protein active sites

The cytochromes P450 (CYPs) play a central role in a variety of important biological oxidations, such as steroid synthesis and the metabolism of xenobiotic compounds, including most drugs. Because CYPs are frequently assayed as drug targets or as anti-targets, tools that provide confirmation of active-site binding and information on binding orientation would be of great utility. Of greatest value are assays that are reasonably high throughput. Other heme proteins, too-such as the nitric oxide synthases (NOSs), with their importance in signaling, regulation of blood pressure, and involvement in the immune response-often display critical roles in the complex functions of many higher organisms, and also require improved assay methods. To this end, we have developed an analog of cyanide, with a (13)CH3-reporter group attached to make methyl isocyanide. We describe the synthesis and use of (13)C-methyl isocyanide as a probe of both bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. The (13)C-methyl isocyanide probe can be used in a relatively high-throughput 1-D experiment to identify binders, but it can also be used to detect structural changes in the active site based on chemical shift changes, and potentially nuclear Overhauser effects between probe and inhibitor.